Production of omega 3 fatty acids by recombinant escherichia coli nissle 1917

ABSTRACT

Described are recombinant  Escherichia coli  Nissle 1917 (“EcN”) cells transformed with genes that express proteins or polypeptides involved in omega 3 fatty acid biosynthesis. The recombinant EcN cells produce eicosapentaenoic acid (EPA) and/or docosahexaenoic acids (DHA). Associated compositions, methods and uses of the recombinant EcN cells are also provided.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/281,865 filed Jan. 22, 2016, the entire contents of which are hereby incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing “25674-P49600US01_SequenceListing.txt” (53,248 bytes), submitted via EFS-WEB and created on Jan. 18, 2017, is herein incorporated by reference.

FIELD

The present disclosure relates to the production of omega 3 fatty acids and more specifically to recombinant Escherichia coli Nissle 1917 for the production of eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA).

BACKGROUND

Eicosapentaenoic acid (EPA, 20:5n3) and docosahexaenoic acid (DHA, 22:6n3) are important omega 3 fatty acids (ω3FAs). In the past decade, EPA and DHA have been promoted as essential dietary components due to their wide ranging physiological effects and their impact on human health. Numerous studies have shown the involvement of EPA and DHA in fetal development (Ramakrishnan et al., 2010), immune function (Yaqoob & Calder, 2007), prevention of Alzheimer's disease (Tully et al., 2003), and protection against cardiovascular disease (Kris-Etherton et al., 2002; Swanson et al., 2012). Omega 3 fatty acids are known as essential fats and currently, fish and fish oil are the main dietary sources for EPA and DHA (Adarme-Vega et al., 2014). The increased awareness of the health benefits of EPA and DHA has led to greater use of fish oils in the pharmaceutical, nutraceutical and agricultural sectors. A higher demand for EPA and DHA will increase the strain on fish populations, which are already in decline, therefore recent investigations have focused on microorganisms as alternative sources of EPA/DHA.

Bacterial biosynthesis of EPA and DHA is limited to a small number of gram-negative marine bacteria such as Shewanella and Colwellia (Bowman et al., 1998). However, these marine bacteria cannot be used for EPA/DHA production due to difficulty in culturing them on an industrial scale. Currently, there is no recombinant source of EPA/DHA available for animal and human consumption. Therefore, in the last decade research has focused on the production of EPA/DHA by recombinant microorganisms such as E. coli, cyanobacteria or by plants (Abbadi et al. 2004; Lopez et al. 2013; Orikasa et al. 2004; Orikasa et al. 2009; Takeyama et al. 1997; Yu et al. 2000). Recently, a gene cluster has been isolated from a marine bacterium, Shewanella baltica MAC1, and cloned in laboratory E. coli strains and Lactococcus lactis subsp. cremoris MG1363 (Amiri-Jami & Griffiths 2010; Amiri-Jami et al., 2014). Sixteen genes were isolated from Shewanella baltica MAC1 of which five (pfaA, pfaB, pfaC, pfaD and pfaE) were shown to be responsible for both EPA and DHA production (Amiri-Jami & Griffiths 2010). Transformation of this gene cluster to different strains of E. coli and Lactococcus lactis MG1363 resulted in production of both EPA and DHA by these bacteria (Amiri-Jami & Griffiths 2010) and production was high in recombinant E. coli strains (Amiri-Jami et al., 2014). However, these strains may not be suitable for commercial production of EPA/DHA.

Escherichia coli Nissle 1917 (EcN) is one of the most studied probiotic bacteria. Probiotic bacteria are living microorganisms that are non-pathogenic and have beneficial effects on human health by improving the microbial balance of the indigenous microbiota (Troge et al., 2012). Several studies have shown that probiotics modulate immune responses (Oelschlaeger, 2010), act as an intestinal barrier (Ohland & Macnaughton, 2010), have an anti-inflammatory potential (Helwig et al., 2006), and are able to secrete bioactive molecules which down-regulate virulence gene expression of bacterial pathogens (Medellin-Pena et al., 2007; Bayoumi & Griffiths, 2012). Probiotic EcN 1917 was isolated by Alfred Nissle in 1917 during the First World War (Nissle, 1918). It is reported that EcN has anti-invasive effects against enteroinvasive bacterial pathogens through a secreted component (microcins) that does not rely on direct physical contact with the invading bacteria or host epithelial cells (Altenhoefer et al., 2004). Moreover, EcN has strong colonization properties and acts as a safe carrier for targeted delivery of recombinant proteins into the host intestinal mucosa (Westendorf et al., 2005). In addition, EcN 1917 has been used for several decades as a probiotic, affording protection against a variety of intestinal disorders such as inflammatory bowel disease (Hernando-Harder et al., 2008).

SUMMARY

Omega 3 fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been reported to have beneficial effects on human health. However, there are relatively few sources of EPA and/or DHA suitable for commercial production. There is remains a need for novel sources of omega 3 fatty acids including those suitable for use in humans.

The naturally occurring probiotic bacterium Escherichia coli Nissle 1917 is unable to produce EPA or DHA. The inventors have surprisingly determined that E. coli Nissle 1917 transformed with a plasmid carrying a EPA/DHA gene cluster isolated from the marine bacterium Shewanella baltica MAC1comprising the genes pfaA, pfaB, pfaC, pfaD and pfaE is capable of producing significant quantities of omega 3 fatty acids. More specifically, as shown in the Examples transgenic E. coli Nissle produced EPA when grown at 10° C. (16.52±1.4 mg g⁻¹ cell dry weight), 15° C. (31.36±0.25 mg g⁻¹ cell dry weight), 20° C. (13.71±2.8 mg g⁻¹ cell dry weight), 25° C. (11.33±0.44 mg g⁻¹ cell dry weight) or 30° C. (0.668±0.073 mg g⁻¹ cell dry weight). Transcriptomic analysis using Reverse Transcription qPCR showed up-regulation of the entire gene cluster in E. coli Nissle. Among EPA/DHA genes, pfaB, pfaC and pfaD were over-expressed (expression ratios of 181.9, 39.86 and 131.61, respectively) as compared to pfaA (expression ratio of 3.40) and pfaE (expression ratio of 4.05). The EPA/DHA-producing probiotic E. coli Nissle may therefore be useful as a source for the commercial production of EPA and/or DHA.

In one embodiment, the recombinant EcN cells described herein may be cultured in bioreactors and EPA/DHA extracted from the culture in order to produce an alternative and safe source for EPA/DHA. Currently, many omega-3 supplements are sourced from fish, which may be retain an unpleasant smell and/or taste or contain lead or mercury contamination. In one embodiment, the EPA/DHA produced using the EcN cells and methods described herein is free of unpleasant smells and/or tastes associated with fish products and is free of lead or mercury contamination. The EPA/DHA produced using the EcN cells and methods described herein may be used in the production of pharmaceutical, nutraceutical, cosmetic and/or agricultural products that contain omega-3 fatty acids.

Accordingly, in one embodiment there is provided a recombinant Escherichia coli Nissle 1917 (EcN) cell or a variant thereof, comprising one or more genes selected from pfaA, pfaB, pfaC, pfaD and pfaE, wherein the cell produces one or more omega 3 fatty acids. In one embodiment, the cell comprises the genes pfaA, pfaB, pfaC, pfaD and pfaE. In one embodiment, the cell comprises genes having sequence identity, such as at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 2 (pfaA), SEQ ID NO: 3 (pfaB), SEQ ID NO: 4 (pfaC), SEQ ID NO: 5 (pfaD) and/or SEQ ID NO: 6 (pfaE). In one embodiment, the cell produces eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA).

In one embodiment, the cell is transformed with a nucleic acid molecule comprising a gene cluster comprising pfaA, pfaB, pfaC, pfaD and pfaE. In one embodiment, the cell comprises a nucleic acid molecule comprising a gene cluster comprising genes having sequence identity, such as at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 2 (pfaA), SEQ ID NO: 3 (pfaB), SEQ ID NO: 4 (pfaC), SEQ ID NO: 5 (pfaD) and SEQ ID NO: 6 (pfaE). In one embodiment, the nucleic acid molecule in a vector. Optionally, the cell is transformed with two or more nucleic acid molecules comprising the same or different genes selected from pfaA, pfaB, pfaC, pfaD and pfaE. In one embodiment, the cell comprises a gene cluster with at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO: 1, wherein the gene cluster comprises pfaA, pfaB, pfaC, pfaD and pfaE.

In one embodiment, the recombinant cell is a recombinant Escherichia coli Nissle 1917 (EcN) cell or a variant thereof. For example, in one embodiment, the EcN cell comprises a genome with at least 80%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to the EcN genome deposited under GenBank under accession no. CAPM00000000. EcN cells suitable for transformation with a nucleic acid molecule encoding the pfaA, pfaB, pfaC, pfaD and/or pfaE genes described herein include DSM 6601 in the German Collection for Microorganisms in Braunschweig, Germany or the probiotic E. coli Nissle bacteria available commercially as Mutaflor™.

The recombinant EcN cells described herein have been demonstrated to produce the omega 3 fatty acids EPA and DHA. For example, in one embodiment, the recombinant cells described herein produce more EPA relative to wild type EcN cells or relative to wild type S. baltica MAC1 cells. In one embodiment, the recombinant EcN cells described herein produce more EPA relative to wild type S. baltica MAC1 cells when cultured at 15° C.

In one embodiment, the recombinant EcN cells exhibit increased gene expression of the pfaA, pfaB, pfaC, pfaD and/or pfaE genes relative to wild type S. baltica MAC1. In one embodiment, the recombinant EcN cell comprises pfaB and expresses a higher level of pfaB relative to the level of pfaB expressed by wild type S. baltica MAC1 when cultured at 15° C.

In one embodiment, the recombinant EcN cells described herein produce omega 3 fatty acids. In one embodiment, the EcN cells produce at least 5, 10, 15, 20, 25 or 30 mg of eicosapentaenoic acid (EPA) per gram of cell dry weight (g⁻¹ of CDW).

In another aspect, there is provided a composition comprising the recombinant EcN cells described herein and a pharmaceutically acceptable carrier. In one embodiment, there is provided a composition comprising the recombinant EcN cells described herein and a culture media. Optionally, the EcN cells are freeze-dried or lyophilized either alone or in a composition. In one embodiment, the composition comprises one or more additional bacterial cells, optionally one or more probiotic bacterial cells or prebiotic chemicals.

In one aspect, there is provided the use of the recombinant cells and/or compositions as described herein as a probiotic and/or nutritional supplement in a subject in need thereof.

Also provided is a method for producing one or more omega 3 fatty acids in vivo comprising administering to the subject the recombinant EcN cells and/or a composition comprising the recombinant EcN cells as described herein. In one embodiment, there is provided a method for producing one or more omega 3 fatty acids in the gastrointestinal tract of a subject. In one embodiment, the omega 3 fatty acid is EPA and/or DHA. In one embodiment, the method comprises orally administering to the subject the recombinant cell or composition.

In another aspect, there is provided a method for the production of omega 3 fatty acids using the recombinant EcN cells described herein. In one embodiment, the omega 3 fatty acid is EPA and/or DHA. In one embodiment, the method comprises culturing one or more recombinant EcN cells of under conditions suitable for the production of omega 3 fatty acids. For example, in one embodiment, the recombinant EcN cells are cultured at a temperature between 5° C. and 30° C. In one embodiment, the recombinant EcN cells are cultured at a temperature between 10° C. and 25° C., between 10° C. and 20° C., between 13° C. and 17° C., or optionally about 15° C. In one embodiment, the method further comprises isolating one or more omega 3 unsaturated fatty acids from the cell culture.

In another aspect, there is provided an isolated nucleic acid molecule comprising a gene cluster comprising pfaA, pfaB, pfaC, pfaD and pfaE. In one embodiment, the nucleic acid molecule is a vector. In one embodiment, the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.

Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will now be described in relation to the drawings in which:

FIG. 1 shows EPA production of gEcN harboring pfBS-PS (pCC1FOS+20 kbp EPA/DHA cluster). Genetically modified E. coli Nissle carrying the 20 kbp gene cluster was cultured in LB broth and grown at 15° C. Total fatty acid was extracted from cells and analyzed as described in the Examples. The data shown are the result of triplicate samples.

FIG. 2 shows a comparison of EPA production by gEcN and S. baltica MAC1 at different temperatures. The gEcN clone was grown at 10° C., 15° C., 20° C., 25° C., 30° C. in triplicates. Total fatty acids was extracted from freeze-dried cells and analyzed by GC as described in the Examples. The data shown are the means±the standard deviations of the result of three independent experiments.

DETAILED DESCRIPTION

Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

As used in the present disclosure, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a recombinant cell” should be understood to present certain aspects with multiple recombinant cells.

The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

The term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.

The term “omega 3 fatty acids” as used herein refers to polyunsaturated fatty acids with a double bond at the third carbon atom from the end of the carbon chain. The term “unsaturated fatty acids” as used herein refers to carboxylic acid compounds containing long aliphatic tail chains in which there is at least one double bond.

The term “DHA” as used herein refers to docosahexaenoic acid, an omega 3 fatty acid. DHA is a carboxylic acid characterized by a chemical structure having a 22-carbon chain containing six cis double bonds.

The term “EPA” as used herein refers to eicosapentaenoic acid, an omega 3 fatty acid. EPA is a carboxylic acid characterized by a chemical structure having a 20-carbon chain containing five cis double bonds.

The term “vector” as used herein refers to a nucleic acid molecule that can be used to transfer genetic material into a host cell where it can be replicated and/or expressed. Optionally, the vector includes an origin of replication, a multicloning site and/or insert, and a selectable marker. For example, in one embodiment the vector may contain an insert comprising one or more genes selected from pfaA, pfaB, pfaC, pfaD and/or pfaE. In one embodiment, the vector contains a gene cluster comprising pfaA, pfaB, pfaC, pfaD and pfaE. Examples of vectors include, but are not limited to, plasmids, bacteriophage, modified viruses (e.g., replication defective retroviruses, adenoviruses or adeno-associated viruses), cosmids, and artificial chromosomes such as bacterial artificial chromosomes (BACs), so long as the vector is compatible with EcN.

The terms “Escherichia coli Nissle 1917” or “EcN” as used herein refer to a non-pathogenic gram-negative probiotic bacteria Escherichia coli strain that is capable of colonizing the human gut. In one embodiment, the EcN cells are of serotype O6:K5:H1. Examples of Escherichia coli Nissle 1917 bacteria include those available as DSM 6601 from the German Collection for Microorganisms in Braunschweig, Germany or commercially as the active component in Mutaflor® (Ardeypharm GmbH, Herdecke, Germany). EcN cells suitable for transformation with a nucleic acid molecule encoding the pfaA, pfaB, pfaC, pfaD and/or pfaE genes are also available from the Canadian Research Institute for Food Safety Culture Collection. Genetic variations of Escherichia coli Nissle 1917 are contemplated in the present disclosure. Accordingly, in one embodiment the recombinant EcN cell comprises a genome with at least 80%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the EcN genome deposited under GenBank under accession no. CAPM00000000. EcN cells may also be identified using the Riboprinter™ microbial characterization system from DuPont. In one embodiment, the EcN cells exhibit at least a 85%, 90%, or 95% match with database entries for EcN cells based on genetic fingerprints.

Nucleic Acid Molecules and Recombinant Cells

In one aspect, there is provided an isolated nucleic acid molecule comprising a gene cluster comprising pfaA, pfaB, pfaC, pfaD and pfaE. In one embodiment, the gene cluster is isolated from S. Baltica (MAC1). In one embodiment, the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In one embodiment, the nucleic acid molecule comprises or consists of the nucleic acid sequence of SEQ ID NO: 1 In one embodiment, the nucleic acid molecule is a vector. In one embodiment, the nucleic acid molecule is a vector comprising an insert comprising or consisting of SEQ ID NO: 1. In one embodiment, transformation of an EcN cell with the vector results in the production of omega 3 unsaturated fatty acids.

In one aspect, the present disclosure is directed towards a recombinant Escherichia coli Nissle 1917 (EcN) cell, or a variant thereof, transformed with a nucleic acid molecule containing one or more genes involved in the biosynthesis of omega 3 fatty acids. As demonstrated in the Examples, the inventors have determined that EcN cells transformed with pfaA, pfaB, pfaC, pfaD and pfaE are useful for the recombinant production of omega 3 fatty acids and in particular EPA.

Accordingly, in one embodiment there provided a recombinant EcN cell or variant thereof comprising pfaA (SEQ ID NO: 2), pfaB (SEQ ID NO: 3), pfaC (SEQ ID NO: 4), pfaD (SEQ ID NO: 5) and pfaE (SEQ ID NO: 6).

Optionally, the recombinant EcN cell comprises one or more genes with sequence identity to pfaA (SEQ ID NO: 2), pfaB (SEQ ID NO: 3), pfaC (SEQ ID NO: 4), pfaD (SEQ ID NO: 5) and/or pfaE (SEQ ID NO: 6). For example, in one embodiment, the cell comprises one or more nucleic acid sequences with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

In one embodiment, the recombinant EcN cell comprises a sequence comprising a gene cluster that includes pfaA, pfaB, pfaC, pfaD and pfaE. As used herein, the term “gene cluster” refers to a continuous linear nucleic acid sequence comprising two or more genes. For example, in one embodiment the recombinant EcN cell comprises a nucleic acid with sequence identity to the gene cluster shown in SEQ ID NO: 1. In one embodiment, the recombinant EcN cell comprises a gene cluster with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1. In one embodiment, the cell comprises a plurality of nucleic acids each encoding for a gene cluster that includes pfaA, pfaB, pfaC, pfaD and pfaE.

Sequence identity can be determined according to sequence alignment methods known in the art. Examples of these methods include computational methods such as those that make use of the BLAST algorithm, available online from the National Center for Biotechnology Information. Sequence identity is most preferably assessed by the algorithm of BLAST version 2.1 advanced search. BLAST is a series of programs that are available, for example, online from the National Institutes of Health. References to BLAST searches are: Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403410; Gish, W. & States, D. J. (1993) “Identification of protein coding regions by database similarity search.” Nature Genet. 3:266272; Madden, T. L., Tatusov, R. L. & Zhang, J. (1996) “Applications of network BLAST server” Meth. Enzymol. 266:131_141; Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI_BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:33893402; Zhang, J. & Madden, T. L. (1997) “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation.” Genome Res. 7:649656.

Percent sequence identity or homology between two sequences is determined by comparing a position in the first sequence with a corresponding position in the second sequence. When the compared positions are occupied by the same nucleotide or amino acid, as the case may be, the two sequences are conserved at that position. The degree of conservation between two sequences is often expressed as a percentage representing the ratio of the number of matching positions in the two sequences to the total number of positions compared.

The genes, gene clusters or variants thereof described herein involved in the biosynthesis of omega 3 fatty acids may be cloned into a vector, transformed into EcN cells and expressed in order to produce omega 3 fatty acids. Techniques for cloning nucleic acid molecules and the transformation and expression of vectors in bacterial cells such as EcN are known in the art. Examples of such techniques are described in Sambrook et al. Molecular Cloning: A Laboratory Manual, 4th edition. Cold Spring Harbor Laboratory Press, 2013, incorporated herein by reference. Nucleic acid molecules containing genes coding for one or more of pfaA, pfaB, pfaC, pfaD and pfaE may be cloned into one or more suitable expression vectors. Suitable expression vectors include, but are not limited to cosmids, plasmids, modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) or artificial chromosomes, so long as the vector is compatible with EcN.

In one embodiment, the vector may also contain a selectable marker gene that facilitates the selection of host cells transformed or transfected with a recombinant molecule of the disclosure. Examples of selectable marker genes are genes encoding a protein which confers resistance to certain drugs, such as G418 and hygromycin.

As used herein, the term “transformation” refers to a process for introducing exogenous nucleic acids into a bacterial cell. The terms “transformed,” “transgenic,” and “recombinant” refer to a host organism such as a bacterial cell, into which an exogenous nucleic acid molecule such as a plasmid or other vector has been introduced. In one embodiment, the recombinant EcN cells described herein may contain multiple copies of the pfaA, pfaB, pfaC, pfaD and/or pfaE genes or associated gene cluster. In one embodiment, the pfaA, pfaB, pfaC, pfaD and/or pfaE genes or associated gene cluster are present in the EcN cell in a copy number greater than 1 such that, on average, each cell contains at least one recombinant vector. In one embodiment, the recombinant EcN cell comprises a recombinant vector comprising multiple copies of each of the one or more genes associated with omega 3 fatty acid biosynthesis.

As used herein, the term “exogenous” refers to a nucleic acid molecule (for example, a circular plasmid DNA sequence), gene or protein that originates from a source foreign to the particular bacterial cell into which it is introduced. An exogenous nucleic acid molecule may be introduced into a bacterial cell either in a stable or transient fashion, in order to produce one or more RNA molecules and/or one or more polypeptide molecules. For example, the genes pfaA, pfaB, pfaC, pfaD and pfaE from S. baltica MAC1 described herein are exogenous when introduced into EcN cells.

The recombinant EcN cells described herein are readily distinguished from wide type EcN cells. In one embodiment, recombinant EcN cells produce detectable levels of omega 3 fatty acids, such as EPA and/or DHA. As shown in FIG. 1, recombinant EcN cells (gEcN) produce significantly more EPA than S. baltica MAC1 cells (SMAC1) or EcN control cells (EcN). Wild type S. baltica cells are available, for example, from the Canadian Research Institute for Food Safety Culture Collection.

As shown in FIG. 2 the inventors have also determined that recombinant EcN cells produce higher levels of omega 3 fatty acids relative to wild type S. baltica MAC1 cells when cultured at a variety of temperatures. For example, recombinant EcN cells produce more EPA relative to S. baltica MAC1 cells when cultured at 10° C., 15° C., 20° C., 25° C. or 30° C. In one embodiment, the recombinant EcN cells described herein produce at least 5, 10, 15, 20, 25 or 30 mg of eicosapentaenoic acid (EPA) per gram of cell dry weight (g⁻¹ of CDW).

Compositions

The present disclosure also provides compositions comprising the recombinant EcN cells as described herein that produce one or more omega 3 fatty acids.

In one embodiment, the compositions comprise recombinant EcN cells and a carrier, optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may be chosen to permit oral administration or administration by any other known route. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (2003—20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. In one embodiment, the compositions described herein include recombinant EcN cells and an animal feed suitable for administration to animals such as poultry.

On this basis, the compositions include, albeit not exclusively, compositions containing the recombinant EcN cells in association with one or more acceptable vehicles, carriers or diluents. These compositions may be supplied, without limitation, as powders, caplets or tablets whereby the bacterial cells are in a dormant but alive state achieved through the use of lyophilization or freeze-drying. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. The lyophilized powder may be reconstituted with sterile water or another vehicle, carrier or diluent prior to administration to the patient. In one embodiment, the composition comprises recombinant EcN cells in association with animal feed, such as an animal feed suitable for farm animals. In one embodiment, the animal feed is for poultry.

Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. In one embodiment, the compositions described herein contain a therapeutically effective amount of recombinant EcN cells that produce omega 3 fatty acids, together with a suitable amount of carrier.

Optionally, the composition may comprise one or more additional probiotic microorganisms such as Lactobacillus or Bifidobacterium. Alternatively or in addition, the composition may comprise one or more prebiotic chemicals. As used herein, the term “prebiotic chemicals” refers to a substance that stimulates the growth of beneficial bacteria such as probiotic bacteria. Prebiotics include, but are not limited to, fermentable carbohydrates such as oligosaccharides, galactans and beta-glucans.

Also provided are cell cultures comprising populations of recombinant EcN cells as described herein. For example, in one embodiment there is provided a cell culture comprising recombinant EcN cells that produce omega 3 fatty acids and culture media. In one embodiment, the culture media contains glucose. In one embodiment, the culture media is LB media or another culture media known in the art to support the growth of EcN cells.

Uses and Methods

In one aspect, the present disclosure also relates to uses of the recombinant EcN cells and/or compositions as described herein.

In one embodiment, the recombinant EcN cells and compositions disclosed herein may be used as a probiotic. As used herein, “probiotic” refers to live, non-pathogenic microorganisms that beneficially affect their animal or human hosts. The administration of probiotics may assist in maintaining the natural balance of microflora in the intestines or may improve the properties of native microflora, or have other beneficial effects such as for the prevention or treatment of disease.

In another embodiment, the recombinant EcN cells and compositions disclosed herein may be used as a nutritional supplement. As used herein, “nutritional supplement” refers to a dietary addition intended to provide nutrients that may otherwise not be consumed in sufficient quantities, such as omega 3 fatty acids. In one embodiment, the recombinant EcN cells and compositions disclosed herein may be used as a nutritional supplement to increase the production of omega 3 fatty acids by an animal, such poultry.

The recombinant EcN cells and compositions disclosed herein may be used in vivo, such as in a subject in need thereof. In one embodiment, the EcN cells and compositions are for oral use. In one embodiment, the subject is a human. In one embodiment, the subject is an animal, such as a mammal. In one embodiment, the subject is a farm animal. In one embodiment, the farm animal is poultry. In one embodiment, the animal is a chicken.

In another embodiment, there is provided a method for producing omega 3 fatty acids in the gastrointestinal tract of a subject comprising administering to the subject a recombinant EcN cell or composition thereof as described herein. In one embodiment, the recombinant EcN cells or composition thereof is orally administered to the subject.

The recombinant EcN cells disclosed herein may be used for the production of omega 3 unsaturated fatty acids such as EPA and/or DHA. In one embodiment, the recombinant EcN cells may be used for the production of EPA.

In one aspect, the present disclosure provides methods for the production of omega 3 fatty acids in vitro. In one embodiment, there is provided a method for the production of an omega 3 fatty acid comprising culturing one or more recombinant EcN cells under conditions suitable for the production of omega 3 unsaturated fatty acids by the cells. As shown in FIG. 2, recombinant EcN cells produce EPA when cultured at a variety of different temperatures. Remarkably, the production of EPA has also been demonstrated to increase at around 15° C. relative to culturing the cells at 10° C. or 30° C.

Accordingly, in one embodiment, the methods described herein include culturing the recombinant EcN cells at a temperature between 5° C. and 30° C., between 10° C. and 25° C. or between 10° C. and 20° C. in order to produce omega 3 fatty acids. In a preferred embodiment, the methods described herein include culturing the recombinant EcN cells at a temperature between 13° C. and 17° C., optionally about 15° C.

The terms “produce” or “production” as used herein refers both to small-scale production and large-scale production of omega 3 unsaturated fatty acids such as EPA and DHA. Both small- and large-scale production may make use of bioreactors or incubators where the recombinant microorganisms are grown to a specific density in a first phase and the production of omega 3 fatty acids occurs in a second phase. Various culture conditions and culture media, such as Luria Bertani (LB) broth, are known in the art to allow for small- and large-scale fermentation of E. coli cultures such as EcN.

In one embodiment, the methods described herein include isolating or separating one or more omega 3 unsaturated fatty acids from the culture. Suitable isolation and separation techniques are well known in the art. For example, the harvested biomass may be centrifuged and dried (e.g. by freeze drying overnight, spray drying, tunnel drying, vacuum drying or other similar methods) and the fatty acids extracted thereafter (e.g. as described previously in Amiri-Jami & Griffiths, 2010).

The omega 3 unsaturated fatty acids produced using the materials and methods described herein may be used in a variety of applications. For example, in one embodiment, the omega 3 unsaturated fatty acids are for use in a nutritional supplement. In one embodiment, the omega 3 unsaturated fatty acids are for use in a cosmetic and/or skin care product. In one embodiment, the omega 3 unsaturated fatty acids produced using the methods described herein are useful for topical application to the skin and/or hair of a subject to improve the look or feel of the skin and/or hair of the subject. In one embodiment, the omega 3 unsaturated fatty acids are for use in an agricultural supplement or animal feed.

The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

EXAMPLES Example 1 Cloning and Characterization of Recombinant EcN Cells for the Production of Omega 3 Fatty Acids Materials and Methods

Strains, plasmids and growth conditions: The bacterial strains and plasmids used in this Example are described in Table 1. EcN 1917 cells obtained from the culture collection of the Canadian Research Institute for Food Safety (CRIFS), University of Guelph, were purified several times by sub-culturing an isolated colony on fresh Luria Bertani (LB) Agar (Difco, Detroit, USA) incubated at 37° C. overnight. An isolated colony of transformant E. coli EPI300T1 was inoculated into a 50 mL sterile tube containing 10 mL LB broth supplemented with 12.5 μg mL-1 chloramphenicol (LBCm) and subsequently incubated at 37° C. overnight while shaking at 300 r.p.m for plasmid isolation. A single colony of purified EcN harboring pfBS-PS, EcN (as negative controls) and S. baltica MAC1 (as positive control) were transferred to 10 mL of LB broth supplemented with 12.5 μg mL-1 chloramphenicol (LBCm), LB broth and Marine broth (MB), respectively and were incubated at 37° C. (E. coli strains) and 30° C. (S. baltica MAC1) overnight. Sterile 250 mL flasks containing 200 mL LBCm, LB and MB were inoculated with 2 mL of each overnight culture and incubated at 10° C. for 8 days, 15° C. for 4 days, 20° C. for 2 days, 25° C. for 1 day or 30° C. for 1 day for fatty acid extraction.

Transformation of E. coli Nissle: E. coli Nissle was transformed with the pfBS-PS plasmid carrying the EPA/DHA gene cluster, previously isolated from a marine bacterium. Plasmid pfBS-PS was isolated from 3 mL overnight E. coli EPI300T1 culture using a QIAprep Spin Miniprep Kit (Qiagen,Toronto, Canada) as described by the manufacturer. The isolated pfBS-PS plasmid was digested with NotI-HF to confirm that the plasmid contained the large 20 kbp EPA/DHA gene cluster. EcN 1917 was transformed with pfBS-PS by electroporation using the GenePulser Xcell Electroporation System (Bio-Rad Laboratories, Mississauga, ON). For electroporation, the pfBS-PS was added to 50 μL of the ice-cold EcN cells and then transferred to an ice-cold 0.2 cM electroporation cuvette. A single pulse was applied at field strength of 2.5 kV, 200Ω resistance and 25 μF capacitance. Immediately, after electroporation, the cell suspension was mixed with 0.95 mL of S.O.C. medium and incubated at 37° C. for 1 h with shaking at 250 r.p.m. Appropriate dilutions of the cells were spread on LB plates containing 12.5 μg mL-1 chloramphenicol. Antibiotic resistant colonies were selected after 18 h incubation at 37° C. To confirm if selected EcN transformants contained the 20 kbp EPA/DHA gene cluster, colony PCR for pfaA and pfaD genes was performed using primers 5A and 8D as described previously (Amiri-Jami & Griffiths, 2010). Isolated plasmids from EcN clones positive for EPA/DHA genes and a negative control (plasmid without insert) were digested with NotI-HF. Digested and undigested plasmids were separated in 1% agarose gel.

Fatty acid analysis: The bacterial cells from 200 mL of EcN transformants, EcN (negative control), and S. baltica MAC1 (positive control) were harvested by centrifugation at 8000 r.p.m for 17 min at 4° C., then freeze-dried overnight. Total lipids from the freeze-dried cells were extracted as described previously (Amiri-Jami & Griffiths, 2010). The fatty acid methyl esters (FAMs) were analyzed using an automated Agilent 6890 Gas Chromatography (GC) system (Agilent, Palo Alto, USA) and a Varian 3800 GC/Saturn2000 ion trap mass spectrometer in external El mode (GC-MS) (Varian, Mississauga, Canada) as described previously (Amiri-Jami & Griffiths, 2010). Compounds were detected by comparison with relative retention time and mass spectra of pure EPA and DHA standards (Sigma Aldrich, Oakville, Canada).

RNA extraction and cDNA transcription: RNA was isolated from transformant EcN 1917 (positive for EPA/DHA production), EcN (negative control) and S. baltica MAC1 (positive control) grown in LBCm, LB and MB, respectively and incubated at 15° C. to an absorbance of 0.35 (A600). The total RNA was extracted using an RNA purification kit (Norgen bioTek, Thorold, ON, Canada) according to the manufacturer's instructions. DNA was eliminated from extracted total RNA using DNase I recombinant RNase-free (Roche Applied Science, Laval, QC, Canada) as follows: 20 μL of total RNA was incubated at 37° C. for 20 min with 10 U of DNase I, 10 U of RNase inhibitor, and 5 μL of incubation buffer in a total volume of 50 μL. The RNA was then purified using the cleanup kit (Qiagen, Ontario, Canada). Purified RNA was transcribed into cDNA using the TruScript™ First Strand cDNA Synthesis Kit (Norgen bioTek, Thorold, ON, Canada) according to the manufacturer's instructions. For control samples, reverse transcriptase was replaced by RNase-free water. The cDNA synthesis was performed in a Gradient Master-cycler (Eppendorf, Mississauga, ON, Canada) under the following conditions: 25° C. for 5 min, 50° C. for 60 min and 70° C. for 15 min. The cDNA was stored at −20° C. until further use.

Quantitative real-time PCR: The expression of EPA/DHA genes (pfaA, pfaB, pfaC, pfaD, pfaE) was determined by RT-qPCR. The primers used for RT-qPCR are described in Table 2. The housekeeping gene 16S rRNA was used for normalization of the expression as described by Pfaffl, 2001.

Real-time PCR was carried out in a ViiA™7 detection system (Applied Biosystems). The reaction mixture contained 1.5 μL cDNA, 1 μL of each primers (final concentration of 600 nM), 10 μL SYBR Select PCR Master Mix (Applied Biosystems, Carlsbad, Calif.), and 6.5 μL of DNase/RNase-free deionized water. The amplification conditions were as follows: 95° C. for 10 min, 40 cycles of 95° C. for 15 sec, annealing/extension at optimal temperature for each pair of primers (55° C. for pfa B-E genes, 58.3° C. for pfaA gene and 60° C. for 16s rRNA gene) for 1 min. The PCR conditions used for the pfaA gene were different from other genes; being 95° C. for 10 min followed by 40 cycles of 94° C. for 30 sec, 58.3° C. for 1 min and 72° C. for 1 min. Thermal cycling, fluorescent data collection, and data analyses were carried out by ViiA™ 7 Software according to the manufacturer's instructions.

Real-time PCR amplification efficiencies (Table 3) were calculated from the slope of the standard curve using the data collected from serial dilutions of the template DNA for each gene according to the equation described by Rasmussen (2001) and Pfaffl (2001):

E=10^([−1/slope])

The relative changes in gene expression were calculated as described previously (Pfaffl, 2001) using the formula:

Ratio=(E target)^(delta Ct target (control−sample))/(E ref)^(delta Ct ref (control−sample))

The cycle threshold (Ct) value is the PCR cycle at which an increase of fluorescence is first detected above the baseline signal. The experiment was performed in duplicate. A relative increase or decrease in transcription value of more than two-fold was considered as significant up- or down-regulation, respectively (Pfaffl, 2001).

Results and Discussion

Recombinant production of EPA/DHA by E. coli Nissle 1917: E. coli Nissle 1917 was transformed with pfBS-PS carrying the 20 kbp EPA/DHA gene cluster. Chloramphenicol-resistant transformants were tested for pfaA and pfaD genes by PCR. Digestion of isolated plasmid DNA from the positive clone (gEcN) for pfaA and pfaD genes with Notl resulted in two bands of size 20 kbp and 8 kbp corresponding to the EPA/DHA gene cluster and plasmid pCC1FOS, respectively. Fatty acid methyl ester analysis of gEcN by GC showed two extra peaks at retention times corresponding to EPA and DHA standards. No extra peaks were detected in the fatty acid profile of the negative control (EcN). The EPA/DHA peaks detected by GC were further analyzed by mass spectrometry. The GC-MS results confirmed that the large extra peak (EPA) detected by GC corresponded to the molecular mass of EPA. However, the small peak identified for DHA by GC was not detected by GC-MS due to its very low concentration.

The transgenic E. coli Nissle produced EPA when grown at 10° C. (16.52±1.4 mg g-1 cell dry weight), 15° C. (31.36±0.25 mg g-1 cell dry weight), 20° C. (13.71±2.8 mg g-1 cell dry weight), 25° C. (11.33±0.44 mg g-1 cell dry weight) or 30° C. (0.668±0.073 mg g-1 cell dry weight). Although DHA was also produced at all these temperatures, it comprised less than 0.2% of total extracted fatty acids. Transcriptomic analysis using Reverse Transcription qPCR showed up-regulation of the entire gene cluster in E. coli Nissle. Among EPA/DHA genes, pfaB, pfaC and pfaD were over-expressed (expression ratio of 181.9, 39.86 and 131.61, respectively) as compared to pfaA (expression ratio of 3.40) and pfaE (expression ratio of 4.05). Accordingly, the EPA/DHA-producing probiotic E. coli Nissle may be used as a safe and economical source for the production of EPA and DHA.

Recombinant E. coli Nissle 1917 produced EPA at all tested temperatures and traces of DHA were observed following growth at 15° C. or 20° C. Transformant gEcN produced 31.36±0.25 mg EPA g-1 of cell dry weight (CDW) at 15° C., which is almost 16 times greater than EPA produced by the marine bacterium S. baltica MAC1 (1.96±0.05 mg EPA g-1 CDW) at the same temperature (FIG. 1). The amount of EPA produced by gEcN ranged from 0.668±0.073 to 31.36±0.25 mg EPA g-1 CDW when it was grown at different temperatures (FIG. 2). The level of EPA produced by gEcN was significantly higher at all temperatures tested compared to the strain from which the genes were originally cloned, S. baltica MAC1.

Transformation of the EPA/DHA gene cluster isolated from different marine bacteria to E. coli strains has been reported (Amiri-Jami et al., 2014; Amiri-Jami & Griffiths 2010; Orikasa et al., 2009; Okuyama et al., 2007). However, there is need for a safe, alternative and economical source for the industrial and pharmaceutical production of EPA and DHA. The EPA/DHA gene cluster has been transferred to Lactococcus lactis subsp. cremoris, a food grade bacterium, however production of EPA and DHA was very low (Amiri-Jami & Griffiths, 2014).

In contrast, when the large 20 kbp EPA/DHA gene cluster was transferred to E. coli Nissle 1917 (EcN), genetically modified EcN was able to produce high levels of EPA and traces of DHA following growth at different temperatures. EcN has been applied as a safe microorganism and a probiotic reagent against intestinal disorders for many decades. The safe human use of modified EcN to deliver nutrients such as beta-carotene (Miller et al., 2013), and proteins like defensin (Seo et al., 2012) and ferritin (Hill et al., 2011) has been reported.

Comparison of EPA/DHA genes Expression in gEcN and S. baltica MAC1: The expression of EPA/DHA genes was studied in gEcN and S. baltica MAC1 grown at 15° C. Wild-type E. coli Nissle 1917 was used as a negative control. The transcription of the EPA/DHA genes (pfaA, pfaB, pfaC, pfaD, pfaE) was investigated and shown to be significantly up-regulated in gEcN compared to S. baltica MAC1 (Table 3). Among all five genes, pfaA had the lowest (ratio of 3.40; Table 3) and pfaB had the highest (ratio of 181.9; Table 3) expression ratio. These results indicate that the meaningful increase in EPA levels by gEcN is due to changes in expression of all corresponding genes when compared to S. baltica MAC1. In addition, genes pfaB, pfaD and pfaC were highly over expressed.

The expression of EPA/DHA genes in gEcN was also compared to that of S. baltica MAC1. In S. baltica, MAC1 genes are located in the genomic DNA, while in gEcN, the genes are plasmid-mediated. These data show that there was a significant increase in expression of all five genes, responsible for the production of EPA/DHA in gEcN compared to S. baltica MAC1. This could be related to the higher copy number of the EPA/DHA genes when they are carried in a plasmid compared to when they are chromosomally mediated (one copy for each gene). Among the five genes, pfaB was highly up-regulated compared to other genes. In addition, it has been reported that pfaB gene encodes beta-hydroxyacyl-ACP dehydratase (HD), beta-ketoacyl-ACP synthase (KS) and acyltransferase (AT) (Amiri-Jami & Griffiths 2010). Allen & Bartlett (2002) have described the role of these domains in the synthesis of EPA in detail. The activities of the HD domains introduce multiple double bonds into a single acyl chain in the absence of desaturation reactions. Moreover, intermediates in the biosynthetic process are presumably bound to acyl carrier protein (ACP) domains as thioesters with AT domains being required for the loading of the starter and extender units while KS domains are involved in condensation reactions. Orikasa et al. (2009) reported that pfaB gene encodes the key enzyme determining the final product in EPA/DHA biosynthesis. This study revealed that up-regulation of pfaB domains might be responsible for higher production of EPA. However, the level of DHA production by gEcN remained low similar to that of S. baltica MAC1. Accordingly, the biosynthesis of DHA could be influenced by pfa gene products other than pfaB. In addition, it is possible that the host factor(s) might have some regulatory functions on the biosynthesis of EPA and DHA.

The present example describes the genetic engineering of a commercial probiotic bacterium with the ability to produce high levels of EPA at different temperatures in a simple medium. Since EcN is a normal and ecologically important inhabitant of the human and animal intestinal tracts, EPA/DHA-producing E. coli Nissle or fat isolated from gEcN may be a cost effective, sustainable and convenient source for industrial production of omega 3 fatty acids for human consumption and pharmaceutical applications. Moreover, recombinant EPA producing E. coli Nissle is also expected to be useful for studying the delivery and production of EPA in the human intestine and its immunostimulatory effects.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosures as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims.

All publications, accession numbers describing biological sequences, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, accession number describing a biological sequence, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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TABLE 1 Bacterial strains and plasmids used in this study Source Description Strain or plasmid CRIFSCC* EPA+, DHA+, marine bacterium S. baltica MAC1 CRIFSCC EPA− , DHA− bacteria E. coli Nissle 1917 CRIFSCC pfBS-PS (pCC1FOS + EpA/DHA E. coli EPI300T1 gene cluster) This study pfBS-PS gEcN pCC1FOS + 20 kbp EPA/DHA PfBS-PS plasmid CRIFSCC cluster *Canadian Research Institute for Food Safety Culture Collection

TABLE 2 Target genes and primers used for RT-qPCR Primer pairs (forward Product Annealing Reference Gene and reverse) and (SEQ ID NO) (bp) temp(° C.) A F: GGTGTSGGYGGTGGTCAR (7) 1800 58.3 (Amiri-Jami R: CTCACCRAARCTRTGRCC (8) & Griffiths, 2010) B F: TCACCAGTTAGAACGTGAAG (9)  110 55 This study R: TAATCGCTTGCTGACTTAGG (10) C F: CGACTGACTATTTGCTGGTA (11)  101 55 This study R: GTCCACAGGAATGTCGTATT (12) D F: GAAATGGCCGATGTGACTAT (13)  131 55 This study R: TCGTAACGGGTGTAGATTTC (14) E F: CCAATCAATAACCAATCGCC (15)  146 55 This study R: GTGTTATCTCGCTATGCTGA (16) 16S rRNA F: CATGCCGCGTGTATGAAGAA (17)   96 60 (Huijsdens housekeeping R: CGGGTAACGTCAATGAGCAAA et al., (18) 2002) Universal 16S F: ACTCCTACGGGAGGCAGCAGT  199 60 rRNA (MAC1) (19) R: GTATTACCGCGGCTGCTGGCAC (20)

TABLE 3 Expression ratio of pfaA-E genes of gEcN normalized to the expression of 16S- rRNA gene and compared to S. baltica MAC1 PCR C_(t) C_(t) Expression Gene Efficiency value/gEcN value/S.MAC1 Ratio pfaA 2.02 32.71 ± 0.35 35.53 ± 0.89 3.40 pfaB 1.8  24.05 ± 0.005 34.19 ± 1.13 181.90 pfaC 1.74 23.14 ± 0.19 31.16 ± 0.32 39.86 pfaD 1.81 24.40 ± 0.54 33.90 ± 1.15 131.61 pfaE 1.83 34.18 ± 0.06  37.75 ± 0.005 4.05 16s rRNA 1.92  4.10 ± 0.06  5.26 ± 0.03 NA* *Not Applicable C_(t): cycle threshold

SEQUENCE LISTING 21Kb EPA/DHA gene cluster SEQ ID NO: 1 CAGAGCACCACTATGCCTTCAAGCCTTCGCCTTGTCTAA AGCGCGTTGAACTCCCGCTGAATGAACAGATATTTAAT ACGATTGGTATTAAACTGGCTGATAACAAGATAACGCGC AGAAAGAATTATGGCTAAACGCAATCGCAGATCTTAGC GGTTTAGGTGAGTTTACTGGCGCGAAGAAAGCACTCTCT TTCGCCGTTAATAATCAGCAAGTAAGCTCTCTATGCTG CTCAACTGCGCGGTTAGCGTTATGGGAAGCGCGGCATTA CCGGCATGCGCTAAGCTAATGGCAAAGCGATACTCCTC GGTCAAACGACCAAAATGCGTTTGCCAATGCAAGGAGTC AGCTAAATCCAGTGTGTGTATTGGGATTTGTAGCGGGA CTTGTAATGGAATATGTGGATAAACATTAAGCATTGAAG CTACGCCGCAGGCTTGCCTCAATCCGGCATCTTGGCAT GATAACTCAAGCGATTCTGCCGCCAATTGCTCAAACGGC TTAAGCTCAAAGGCAAAGGATTTGAGCGACTGCGCTAA ACCTAAACCAGTCGCCTTAATATAGGACTCCTTTAGCGC CCATAAATCGAAAAAGCGCTGTCGCTGACTCGCTTCAT CGGCGAGAGCGAGCAAGGCTGCAGTTTCTTGGGGAGAAA AATAGTGATTTAGGATTGGATAGATATCCGTCTTCGGC CGCGACCTTTCAATATCCACCCCAAATAAACCAGACTCA ACGCCATCAAACTGAGCCACACCAATCAATAACCAATC GCCGCTATGGCTTAGGTTAAACTCGAGACCAGTTTGCTG CCCTAATGTCGCCGTTAAACTCGGTTTGCCCTTAGCGC CATATTCAAAACACCACTCATGTGGCGATAAATCAGCAT AGCGAGATAACACGGTCCGTAGCGCCGCCCTCACCTGT AACCCCTTTATTTGCACCTTGGGATCGCGATAACGCCTG ACCTTTGCGAGTTCATCATCACTTAACCAAGACTCGGC AAGTGATGTTTGCGCCGCTGAAATATCGTTCAGTGGAAT AAAAAATAGATCCACATTCACGCCGTTTTTGCTAGATG TTAGAGAGTCGGTCATTCGAGAGCCTAGCTGTACAAAGC CCTGCGGCTGATGAGTTATCATTGGCTGGCTTTTTGGA GAGCAGTTAGCAACATTCTTATCTGGTTCATTATTGTCT AGGTTCATCATTATCTGAGTTCATTATGCATCTTAAAT AGTTCCAACTAAGCCCTATTTCAATTCTTAGCCGCACTC TAATAAGAGCAATTCTTAGAACGACTATTAACTCTATC CTAAGCGCAAAATTTTGCTGCAATTGCGCCAAGGTTCCC CCCCAAAAAAAATTAGCAGGATTAGCCACACAGCCTCA TTCATGACTAAAGGGACGCAAGCCAATATTGGGCTATTT TACCCCGATAACTCAAAAAGCTGCCTTCATCGCCGCTA CAAATATAAATCTTCCACTATCAAAGTATTAAACAGCCC AAGCGGCCAGCGAAAACCCAACATAACGCACAGAAAAT ACCATTACTCAACGAAAGCCAATAAATTCCAGTCCCAGA TGATTGTATCGACCCGAAACCTCAGGTAATCTGCATCC CCATTAAGTGACACAGATGATAAGCACAGGTTTTAAGAT GAGTGTCACGACATTAGTCATAAATGTCAGTCGTGACT CAGCCAAAATCGCTTATCAGTCCCAATAGATTATGTCGG ATGATAATTATTTTTAATGGCAACTCGCGACTCTCAAT CTAAAGCGCCGTCATACGCCCCCAAGAGCACGACTCCAG TGGCGCAAAAAGACAGCACACCTCGGCATCGCAACGCC ACCACTACGCCTGAAATGCGACTCTTTATCCAACAATCA GATCTGAGCGTGAGCCAGCTGGCAAAGATTTTAAATAT TACCGAGGCAACGGTGCGTAAGTGGCGTAAGCGTGAGTC CATCAGCAACAGTTCGAACACGCCCCACCATCTCAACA CGACGCTCACGCCGATGGAAGAATACGTGGTGGTGGGAT TACGTTACCAACTCAGACTCACCTTAGATAGATTGCTC CACGTCACCCAAACCTACATTAATCCCAATGTGTCGCGC TCCGGCCTTGCCCGTTGCTTAAAGCGCTACGGCATATC GCGGCTCGATGAGTTTGAAGCACCACAAGTACCCGAACG CTATTTTAATCAATTACCTGTGACTCAAGGCAGCGATA TCCAAACCTATACGGTCAACCCCGAAACCTTGGCCAAAG CCCTCGCATTGCCGAGTACCGACGGCAATACTGTGGTG CAAGTGGTATCACTCACCATACCGCCGCAGCTTACAGAG CAAGCTCCAAGCTCAGTGCTATTAGGCATAGATACGGC AAGTGATTGGATTTACCTCGATATTTATCAAGACAGCAA TACTCAAGCGACCAATAGATATATCGCCTATGTCCTCC GACATGGGCCGTTTCATTTACGAAAGTTGCTCGTTCGCA ACTATCACACCTTCTTAGCCCGTTTTCCCGGTGCCCAT GGCACGCCAAAGACAAACGCGGCTGAACCCCAAAACAAG GTCACCGTATCCAGGTCGACCCGTGGAGACTCTTTATG AGCCATACCCCTTCTGTACCTAATTCTGCGACTGAGTCA AAAAAAGATAAACGACTCAACAAACGTTTGAAAGATAT GCCCGTTGCCATCGTCGGCATGGCCAGTATCTTCGCCAA CTCGCGTTATTTAAATAAATTTTGGGATTTGATCAGCG AAAAAATCGATGCCATTACTGACATCCCAGACACCCATT GGCGCGCCGAAGATTACTACGATGCAGACAAAAGCAAA GCCGATAAAAGCTACTGTAAACGCGGTGGTTTTTTGCCT GAAGTCGACTTCAACCCAATGGAATTTGGTCTGCCGCC CAATATTCTTGAGCTAACAGATACCTCGCAACTGCTGTC GCTGATTGTCGCCAAAGAAGTGCTCGCCGATGCCAATC TGCCCACCGATTATGATCGCGATCGTATCGGCATCACGC TAGGCGTGGGCGGTGGTCAAAAAATCAGTCAGAGCTTG AACTCGCGCCTGCAATATCCAGTATTGAAAAAAGTATTC AAAAGCAGCGGCCTGAGCGATGAAGACAGCGAAATGCT GATCAAAAAGTTCCAAGACCAATATATCCACTGGGAAGA AAACTCTTTCCCAGGGTCCTTAGGCAACGTGATTGCAG GCCGTATCGCCAACCGTTTTGATTTTGGCGGCATGAACT GTGTGGTCGATGCTGCCTGCGCGGGCTCACTTGCCGCT ATGCGTATGGCGTTGACTGAACTGACCGAAGGTCGCAGC GACATGATGATCACCGGCGGCGTCTGTACCGACAACTC GCCGTCCATGTATATGAGCTTCTCAAAAACGCCTGCCTT CACTACCAATGAAACCATTCAACCCTTTGATATCGATT CAAAGGGCATGATGATCGGCGAAGGTATTGGCATGGTAG CACTTAAGCGCCTTGAAGATGCCGAGCGCGATGGCGAC CGGATTTATGCCGTCATCAAAGGCGTTGGCGCCTCATCG GACGGTAAATTTAAGAGTATTTATGCGCCGCGCCCTGA AGGCCAAGCTAAAGCATTGGAGCGCGCCTACGACGACGC GGGTTTTGCCCCGCACAGCATTGGCTTAGTTGAAGCCC ATGGCACGGGCACTGCCGCAGGTGATGTGGCCGAATTCA ATGGCTTAAAATCGGTATTTGCCCAAGGCAACGACACC AATCAACATATCGCGTTAGGTTCAGTGAAATCCCAAGTG GGCCACACTAAATCAACCGCAGGTACTGCTGGGGTAAT CAAAGCCGCGCTGGCGCTGCACCACAAGGTATTACCTGC GACCATTAACGTCAGCAAGCCTAATCCAAAACTGAATA TCGAAAGCTCACCATTCTATTTAAATACCGAAACGCGCC CTTGGCTGCAACGCACTGACGGTACGCCGCGCCGTGCT GGCATAAGTTCCTTTGGTTTTGGCGGCACTAACTTCCAT CTCGTATTAGAAGAATACAAACCCGAGCACAGCCGTGA CGAGCAATATCGTCAGCGCAGTGTGCCGCAAACGCTATT ATTTGCCGCCGCCAATAAAGCCGCGCTGCTTAGCGAGT TAAAAGCTGCGCTGAGCCAAAGCTTGAATACGAACGCGA ATAAGAGCAGCGCTGCGAGCCTGAACGCTATCGCTCAG CAATATCCGCTACGCGCGCTGGCAGACACAGATGCCCGT TTAGGTTTTGTGGCTAAGGATATCGCTCAGCTGCAAGC TCAGTTGAATCAAGCCATCTCTCATCTAGAAAGCAGTGC GCATTTAGAAAGCGGGCTCCTAGATACCAGTGCCAGCG AGGCGTGGCAGTTACCTTCTGGGATCAACTACCGCGCAA AAGCTTTAGTTGCTAAAGATGAATCAAAGAAAGTGGCC GCTCTGTTTGCCGGTCAAGGTTCACAGTACCTGAACATG GGCCGCGAACTCGCCTGCCATTTCCCTGAAATGCGCCA ACAAGTGATGGCCAGCGATAAGGTATTTGCTCACCACGG CCAAACGCCGTTATCCAACATCCTTTATCCTATTCCGG CATTCGATGCCGATGCGGTTAAAGCCCAAGAAGCGGCGC TGACCAATACCCTGTTCGCCCAAAGCGCCATAGGCGCG GTTTCAATGGCGCAATATTCACTATTAACTCAGGCAGGT TTTGCCCCAGATATGGTGGCGGGTCACAGCTTTGGTGA ATTGTCAGCCCTTTGCGCGGCTGGCGTGATTTCGAGTGT TGATTACGTCGAACTGGCCTTCGCCCGTGGGCACGCCA TGGCGCAAGTGCCAAGTGATACTGACGCTCAAGTCGATT TAGGCAAGATGTTTGCCATCATCCTCAAACAAAAGAGT GATATCGATGCGCTTAATCGCTGTTTAGCTCAGTTTGAT GGCGTTAAAATTGCCAACTACAACGCGCCGACACAATT AGTCATCGCAGGTGGCACAGAGCAAACTCAGCTGGCTGC CAAAGCTATTAGCGAACAGGGCTTTAAAGCGATTGCCC TGCCCGTCTCTGGCGCGTTCCACACCCCCTTGGTTGGGC ACGCACAAAAGCCATTTGCCAAGGCCATTGATAAAGCT AAATTCAGCGCGCCTTCAATCGCACTTTATGCCAACGGC ACAGGTCAATTGCACCCAAGCGATGGCAAAGCGATTAA AGCAGAATTCAAACAACACATGCTGCAATCGGTTCGTTT TAGCGAACAACTGCAGGCCATGTATGATGCTGGCGCCC GTGTGTTTGTTGAGTTTGGCCCTAAGAACATACTACAAA AGCTCGTTGAAAATACCTTAAGTGAACACTTAAATGAG CTTTGCCTTGTCAGCATGAACCCGAACCCTAAGGGCGAC AGTGACAGTCAATTACGCTTAGCCGCTGTGCAACTCGC GGTAGCCGGTGTGGCGTTAACTGAGGTTGACCCCTATCA AGCCGTCACGTCACAGGAGATTGCCGAGCGTGAAGCGC CATCGGCGATGAACATCAAACTCACCGCCACTAACCACA TCAGTGCCGCAACCCAAGCTAAGATGGCGAAATCTCTC GCCACAGGCAGCGTGACTAGCCAAGTGCAATATGTGGAT CGCATCGTTGAAACGATTCTCGAAAAAGAAGTCGAAAA AATCGTTCAGAAAGAAGTCATAGTCGAAAAAGTGGTTGA GAAAATAATAGAAGTGGAAGCGAATCAAGTGGCAGCTG TTGAAATGAAACAAAAACTCCTAAGCGTGACGCAAGGCT TAAATCACCAACAAGCGACGGCGCAGATGAGCCCAAGC ACAGCAAACGTCAGCGGCGATGCGTTAACGGCATTTTTC AGCGCACAATCTCAAGCGGCGCAGTTACATCAACAGTT TTTAGCCATCCCGCAGCAATATGGCGATACTTTCACGAC ACTGATGACAGAACAAGCCAAGATGGCGAGCCTTGGCA TTGCAATCCCAGAAAGCCTGCAACGCTCGATGGAAATGT TCCACCAGCATCAAGCGCAAACTCTGCAAAGCCACGCC GAATTTATGCAACTGCAAAGCAGTAGCAGCCAAGCGGCC TTGGCCATGTTAAACAATGCGCCGATTAACTTTACCCC AGCGGTTGCAAGTCAGCCACAAGCAACTGTACCTGTTGC TCCTGCACCTGTTGCTGCATCGACAGTGGCACACAATG CCGCACCAGTCGCGGCTCAGGCTGTTGCGACAAGACCAG CGGTTAGCACACCCGTTCCTCCTGTTGTGCAAACCGCT CCCGTTGCTTATGCGCCTGCTGCGACAGTACAAGTTGCG CCAGCAGCTCCTGCCGCACCCGCATTAGTCATGCCTGC TGTGGTGATGCCAGAAGTGACACCTGTGGCACCAACAAT GAACGGTTTAAGTGCTGAAAAAGTACAGCAAACAATGA TGGCTGTGGTGGCTGGCAAGACAGGCTATCCGACTGAAA TGCTGGAGCTCAGCATGGATATGGAAGCCGATCTCGGT ATCGATTCAATTAAGCGCGTTGAAATCTTAGGCACAGTG CAAGACGAGCTGCCGAGTTTACCTGAACTCAGCCCAGA AGATTTGGCTGAGTGCCGTACCTTGGGTGAAATCGTAAG CTATATGAATAGCAAATTACCTGCAGCGGGAACTATTG CTTCGACAGCAGCGCCAGTTGCTCAAACCATTGCCGCTC CTGCATCTAACGCTTTAAGCGCTGCATTAGTTCAACAA ACTATGATGGCTGTGGTGGCCGACAAGACAGGCTACCCC ACTGAAATGCTGGAACTTGGCATGGATATGGAAGCCGA TTTAGGTATCGATTCTATTAAGCGCGTTGAGATTTTAGG CACAGTGCAAGACGAACTGCCGGGTTTACCCGAACTTA GCCCAGAAGATTTGGCCGAATGTCGCACCTTAGGTGAAA TCGTTACCTATATGAACTCTAAACTGCCAGCAGCGGGT TCAGTTGTAGTTTCTACAACAGCACAAGCCGCGCCTGCC GACAGCGGTTTAAGCGCTGAGTTAGTGCATTCTTCTGA AATCGAGAGCACCATGATGGCTGTGGTGGCCGACAAGAC AGGCTATCCGACTGAAATGCTAGAACTGAGCATGGATA TGGAAGCCGATCTCGGTATCGACTCCATCAAGCGCGTTG AAATCCTAGGCACAGTGCAAGACGAACTGCCGGGCTTA CCTGAACTTAGCCCAGAAGATTTGGCCGAGTGTCGTACC TTGGGTGAAATCGTTAGCTATATGAATAGCAAATTACC AGCAGCAGGAGTTATTGCTTCGACAGCTGCGCCAGTTGC TCAAACCATTGCAGCTCCTGCCGCAATCGGTTTAAGCG CTGCATTAGTGCAACAAACTATGATGGCTGTGGTGGCTG ACAAGACAGGTTACCCCACTGAAATGCTGGAACTCAGC ATGGATATGGAAGCCGATTTAGGTATCGATTCTATCAAG CGCGTTGAAATTTTAGGCACAGTGCAAGACGAACTTCC TGGCTTACCTGAACTCAATCCTGAAGATCTAGCCGAGTG TCGCACCTTAGGTGAAATCGTTGACTACATGAACAGCA AACTACCTGCGGTTGGCTCGACTTCAACTATAACAGCAG CACAGGTCACTGTTACCGCTACAATAAATAACGGCTTA AGTGCTGAAAAAATCCAACAAACCATGATGTCAGTAGTG GCCGACAAGACGGGCTACCCCACTGAAATGCTGGAGCT CAGCATGGATATGGAAGCCGATTTAGGTATCGATTCTAT CAAGCGCGTTGAAATCCTAGGCACAGTACAAGACGAAC TGCCGGGTTTACCTGAGCTAAATCCTGAAGATCTGGCCG AGTGTCGCACCTTAGGTGAAATCGTTATGCTCTTTTCG CAAGATTTAGGGCAAGGCAAGCTAGACCAGAATAATGCG GCGAATGTGGTAAACGTCGCTGTCGCAGTTGAACCGAC TATCGATTTGCTACCCCATGATTTACCACAACATGATTT ACCACCACACAGTGAGGTGGTGCTAAAAAAGTTGCCAG CGGCGGCTGAGCTAACGCAACTATCGCCACAGCAATCGT CAAAGCAATCAGCGCAACAAGCTCAAACACGCGTTTTT GCTAAAGATGCCTGCATTATTATTAGCGATGATGGTCAC AATGCTGGCGTACTTGCCGAGAAATTGCATGCTCAAGG TCTAACGGTTGCGGTTGTGCGCTCGCCAGAAAGCCTTGT AGCCAGTGCATCACCGCTCAATAGCCACATTGCCAGCT TCACATTGGCGGCGATTGACGATATCAGCATCAGCGTAG TAATTAATGAGATCAAAACCTTAGGTCAAGTTGCAGGC TTTATTCATCTGCAACCACAACATAAAACCTCAGCCGAT GCGAAAGGTTTAGTGTTGTCCAGTGCCGCCAAAGCGTC GGTCGAGCAAGCGTTCTTGTTCGCCAAGCACTTACAGCC GCTTTTAACAACAGCAGCAATCGCCAATACTGGCAGCA GCTTTATCAGCGTCAGTCGTATCGACGGTGGTTTTGGTT ATCTTAACCACAGCCAAATTGCCCGCAGCGAGTTAAAC CAAGCGGCATTGGCAGGTCTGACTAAAACCTTGAGCCAC GAGTGGCCAAGCGTGCATTGCCGCGCCTTAGATATCGC GCCAGCACTTGATGCCAAGCAACTGGCCAATGCGGTCAT AGCTGAGTTATTCGCAACCGATAAGCTTTTAGAAGTCG GAGTGAGTGAAAGTGGTGTGAGTGAAGCTGGCGCAACTG AAACGCTGGCACGCGTGACCTTAGTTGCAGGCAAGGCA GATACCCGCCACGGCGCAGCTAACTTGACCAGTGCTGAT AAAATTCTAGTCACTGGCGGCGCAAAAGGCGTGACCTT TGAATGTGCCTTGAGTCTGGCAAAACGCAGCAAGGCGCA CTTTATCCTTGCCGGCAGAAGCAGCCAACAAACCATTC CCGCTTGGGCACAAGGTAAAAATAACAGCGAACTTAAAG CCGCAGCTATTGCCCATATTCAAAACTTGGGCGAGAAA CCTACACCAAAACAAGTAGATGCCTTAGTATGGCCAGTT CAAAGCAGCCTAGAAATTGCAGCCGCACTAGAAGCCTT TACTGCAGTAGGCGCGAGTGCCGAGTATTTAAGTCTCGA TGTAAACAATCCAGACGCGATCGCCAGCACGATAGTGC CGATCACCGAGTTATCACCTATTACCGGCATCATCCATG GTGCGGGCGTACTCGCCGATAAACACATCCAAGACAAA ACCTTAGATGAGTTAGCACGGGTGTATGGCACTAAGGTC ACAGGCATTAGCAATCTGCTTGCCGCACTGGATTTAGA TAAAGTAAAACTGATTGCCCTGTTCTCTTCGGCGGCGGG TTTTTACGGTAATACCGGCCAAAGCGATTACGCCATGT CCAACGACATACTCAACAAAGCCGCACTGCAACTGGCGC AGCAACTGCCAAATGCCAAAGTGATGAGCTTCGATTGG GGTCCGTGGGACGGCGGCATGGTGAATCCTGCGCTGAAA AAGATGTTTATGGATCGCGGTGTTTATGTAATCCCGCT AAAAGCAGGTGCTGAGTTATTTGCGAGCCAATTACTGAG CAACACAGGCGCACAATTGCTGGTCGGTACTGACATGC AAGGCAGCGCACCTCATGACGACACGCCTAACGAAGTAC AAGAAACTGAAGGTAGTAATCTAAAAAAGCCTGAAGCG GATCTGACCACTGACTCGTCGGATCCGCATGCTTTGCTT AACGCTTTAAATCCAAGCGCAGTAAATATAAGCGCTGT AAAACTGCAGCGCACACTGGACCCTAAGGCAATGATCTT TATTGAAGATCACTGCATTAACGGTAATCCCGTATTAC CGACTGTGTGCGCGATTCAGTGGATGCGTGAAGCCGCCT TTGATGTGCTCAAGCAACCAGTCAAAGTGCAAAGCTAC AAGCTGTTGAAGGGCATTATTTTCGACGCTATGACGTTA GAAAACGGCGCGCCCATCACGCTAGAACTTGAGCTTGC ACCGATTGCGTTAACGGATAAAGCCGCGAAAGACACAGA CGAGTACTTGAGTGGACAATTCAGTGCCTTAATCAGCT TTGAAGGTCGTCCGCAATATCAAGCCATCTTAGTGATTG ATGATGCTCCTAGTGATAACTTAGCCACTAATAGCAAA GCAACTGCCTTTGACGCGCATAGCTTGGCAGGATTTTCT GCCATCACAACCGCAAGCAGCCTTTACAGCGACGGCAC GCTTTTCCACGGCCCAAGACTGCAAGGTATTGAGTCTGT GGTGAAGTTTGACGATGCGAGCTTAGTCGCCAAGGTGA GTCTTCCCCATGTTGCGTTAGCGGACTGCGGGAGTTTTG TGCCCAATCTGGCCCCTAAAGGCTCACAAGCTTTTGCA GAAGATTTGTTGCTGCAGGCGATGCTGGTATGGGCAAGA CTCAAATATGGCGCGGCGAGTTTGCCATCAAGCATTGG TGAGTTTATCTCCCATGCGCCGTTTGCCTTTGGCGATAC AGGGTATCTAGTGCTCGAGGTGGTAAAACACTCTGGCC GTGCCCTTGAAGCCAATATTGCACTTTATCATCAAGATG GCCGCTTGAGCTGCGAGATGAACAACGCCAAAGTCACC ATCAGCAAAAACCTTAACGGTGCATTCTTAGCGAATAAA GTTGCTAATAAAAGCATTGAAAGCGTGGAGGCAAAAGT CGAGTGAACTTAGCCTATCCTTTAGCAATGACCCATAAC GGCCAAGCTGAAACGTCGATAGCTGACAAGGCTATTGC TGACAAAAGCGCCAAGCCACTGCGTATTGCAGTGTTGCT TGGCGATGCGGTCAACCTTGACTCTCACTCAGCCCAAG TATTAGGGACGTTTACTGAACGTGAAAGAGTTCAAATTT GCGCTGCCGATGCCAACCAATCGACAGTACATAGGCCG ACAGCTCATGAACCCAAAGAGCATGAACCGTCAGTGTAT GAGCAAAGGTCGCTCACAGCCTTGTTAGGCCAAGCAAC AACAGCCATTGAGCAAGGCAAGCTCGTTGAACTTAAATT TGAGGATGGTAATCTACCCCAGTATCTGTATTTGCTCG ATGGTTTACGCGCCGCCAAACTGCGTCTTCACGCCCACG CGTTTATTGCTGGCTTTGCCGCTGGCAATGAGGTTACA GATGTTGCAAATGCGGCGACTGTTGCAAATAAGGCGTTA GCGGCAGCAAAGCGCAGCCCAGCTCAAACCGTTCAACA TCAGACTGTCGCCAACACACTCAACGAGGCATTTGTTGC GCTTCGCCAAGGCGTAACCGCCCTCGCCGCGCGAACAC AAGCGCCGCTTAAAGGCACTACTGGTATAAAACAGACAA ACGACACCAATCATCAAACTGGCTATTGGTTTAGCGAC CAACATCAAGCGCGGGTGTTGTGTCTAAATCTTGTAGCA AAGACGTCACATCAAGCGGATGAGTCTCGAAATCTAAG CCAAAGCCTAGTGCTGACCCAAGGCACACAACTCGCCGC GCCCAAAGCCCTTGTCGATGAAAACCGGCTGTTTGTGC CGATAAGTAGTGACAGTATTAATGAGTTAAAAGCAAAGC TGTTTCAGTTGCTTAGTTCACTAGACATTGGCGCACCA GACACGTCATCTGCATCACATCAACTCACATTCTGGTTT GAACGCTACGATGCCAACGCACCACTGGCATTAGTCTT GATGGCGGCATCAAGTGATGATCTCAAACTTGAAGCCAA AGCCATGCTTACGGCGCTTGAAAATGATGCTATTCGTC ATCATGGTCAACACTTTAAGACACCAGCGGGTAGCTGCT TTACCGCTAAGCCGCTAGGGGATGCGGGACTGACTTTT GTGTATCCTGGCGTTGGCACGGTTTACGCCAATATGTTC AATAACTTGCATGAGTATTTCCCCGCGCTGTATCACCA GTTAGAACGTGAAGGCGATTTAAGCGCCATGCTGCAATC ACCGCAGATTTATGCGGCAGATGTTAAAACCGCGGCTG GCATGAACCTAAGTCAGCAAGCGATTAGTGGCGTGGGGG CCAGTTATCTATTCACTAAACTGTTGACCCAAGTCTTT AATATTAAGCCTAAAATGGCGCTCGGTTACTCAATGGGC GAAGCAGCCATGTGGGCCAGCCTAGATGTGTGGCAAAC ACCGCACGCTATGATTAACGCCACTGAAAATAGCGATAT TTTCAACCATGCGATTTCTGGTGAGCTAACTGCTGTGC GCCGAGCATGGCAGCTTGCGGATGATGAAGCCATAGTGT GGAACAGCTTTGTGGTGCGCGCTGATAGCCATGAAATC AAGGCATTATTGCCAGAGTTTCCTCGCGCCTACTTAGCC ATCACCCAAGGTGATACTTGTGTCATTGCAGGCTGCGA AACAAGCTGTAAAGCCCTGCTTGCCACGTTAGGCAAACG CGGGATTGCCGCCAATCGCGTCACGGCAATGCATACCG CGCCTGCCCTGTTAGTCCATGGGCAAGTACAAGATTTCT ATACTCAAGCGCTAAAACCTGAGGCACTGGAACCTGAT GCGCTGAAAGCGGCAGCGCAAGATTCGTCTGTTCGCTTT ATTAGCGCAGCGCAAACTGCGCCAGTAATCGTGGATAG CCACAGCATTGGCCGCGCGATTGCCGATACCTTTTGTTC GCCACTCGACTTTAGTGCGCTCATTCAAAATGCCACTG AGCAAGGCGCAAGGCTGTTTGTCGAAGTGGGCGCCGATA GGCAAACCAGTACACTCATAGATAAAATCAGCCATGCC CACGCAAGCCAAAGCTCTGCGAACGCGGCGACAGCCGCC ATTGCCTGCAATGCCAAAGGTGCCGACGCGATCACCAG CTTACTCAAGTGTTTAGCTCAGCTAATAAGCCACAGAGT GCCGCTTTCGCTCACGCCGCTTATTCAGCCATTAAGTG CTAACGCAGCCCCTTTATCATCAGCAGTATCACCAAAAG GAGAACCCCAGTGAGTTCTCAGCATTCCCCTACGATTG ATAAAACTACCGTGCCTACCATTGCGTCAAACCGCGCAT CAAAAAGCGCGTCAAAAATTGCGATCGTCGGCCTCGCG ACTCAGTATCCTGACGCCGATAATCCTCAAACCTTTTGG CAAAATCTGCTGGATAAAAAAGACTCTCGCAGTCAAAT TAGCCGCGAGAAGCTCAATGCCAATCCCGCCGATTACCA AGGTGTGCAGGGTCAATCTGACCGTTTTTACTGTGATA AAGGCGGCTACATCCAACACTTCCAGTTTGATGCCAAGG GTTATCAACTGCCTGAGTCCGCCTTTGACGGTTTAGAT GAAAGCTTTTTATGGGCACTCGATTGCAGTCGCAAAGCC CTCCAAGACGCAGGGATTGCCCCAAGCGATGCCGTACT GGCGCGCACAGGTATCGTGATGGGAACCTTGTCGTTCCC CACAGCCCGCTCCAACGAATTATTTTTGCCTCTGTATC ATCAAACCGTTGAAAAGGCGCTGCAAAACAAACTGAATC AAAGCACTTTTCAGCTGGCTGATTTTAACCAAGCTCAC GCCGACAAAGCATTAAACGTCGAGCAAGCATTAAACGTC GCCAATGGCGCCGTTGCCCACACAGCTTCAAAGCTAGT CAGCGATGCACTCGGTTTAGGTGGCACTCAGTTAAGCCT GGACGCTGCCTGCGCTAGCTCGGTTTACGCACTTAAAC TCGCCTGCGATTACCTGACCACAGGCAAGGCCGATATGA TGCTTGCTGGCGCGGTATCGGGCGCAGATCCCTTCTTT ATCAACATGGGATTCTCGATTTTTCACGCCTATCCTGAT CATGGAATCTCAGCACCCTTTGATAGCAACAGCAAGGG CTTATTCGCCGGAGAAGGCGCAGGGGTGTTAGTATTAAA ACGCTTAGAAGATGCCGAGCGCGATGGCGATAACATCT ATGCCGTCGTCAGCGGCATTGGTTTGTCGAACGACGGTA AAGGCCAGTTTGTATTAAGCCCCAACAGCAAGGGCCAA GTGCAAGCTTTCGAGCGCGCCTATAGTGCGGCAAACACA CTGCCCGCCAATATCGAAGTGATTGAATGCCACGCCAC CGGCACGCCGCTTGGGGATAAGGTCGAACTCGCCTCGAT GGAACGTTTCTTCGAGGACAAACTCGCGGGCTCTGCAG TGCCGCTGATCGGTTCGGCAAAATCCAACTTAGGCCATT TGCTCACAGCCGCAGGCATGCCGGGGATCATGAAGATG ATTTTTGCCATGCGCTCGGGTCGACTGCCGCCAAGTATT AACTTATCAGCGCCGATATCCTCGCCTAAGGGCTTGTT TAGCGAAAAGAATCTGCCAACAGAATTACATGCTTGGCC CGATAAAGCCGGAAACTCCCGCCGCCACGCCGGTGTTT CCGTGTTTGGTTTTGGCGGCTGTAACGCGCATTTGTTGC TGGAATCCTATGTTGCCAATACAAACAAAAAGAATGAA CAAGCCGCAGCTGCTGTAAGTTATCAGCACACGCCATTA AATATCATTGGCTTAGCGTCGCACTTCGGCCCTTTATC CTCCATTAATGCACTGGATAGCACGATTCAAGCTCGGCA ACATGCCTTTATCCCGCTGCCCGCTAAACGCTGGAAAG GCTTAGACAAACACCCTGATATTCTGGCCAACGTTGGTT TAAGCGGAACTGGCCTCGCAGCCGCACCACAGGGCGCG TATATCGACCAGTTCGATTTTGACTTCCTGCGTTTTAAA GTGCCGCCCAATGAAGATGACCGCCTGATCTCCCAGCA ACTGCTGTTGATCAAAGTGGCAGATGAAGCGATTCGTGA TGCCAACCTTAAGCCCGGTGGCAAGGTTGCTGTATTAG TGGCGATGGAAACTGAACTCGAATTACATCAGTTCCGTG GCCGCGTAAATCTGCACACGCAACTGGCAGACAGTCTT AAAAAGCAAGGGATCACCCTCACACAAGCCGAGTATCTC GCCCTTGAAAAAATCGCCATGGACAGTGTGCTCGATGC CGCCAAGCTGAACCAATACACCAGTTTTATTGGCAACAT CATGGCGTCACGCATCGCATCCCTTTGGGACTTTAATG GCCCCGCCTTTACCATTTCGGCGGCGGAACAATCGGTCG CCCGCTGTATCGATGTGGCCGAAAACCTCTTGTCCCAA GAATCCTTAGATGCCGTAGTGATCGCCGCCGTCGATTTA AGTGGCAGTCTAGAGCAAGTGATCCTCAAAAACGCCGT ATCACCAGTGGCATTTAATGCCACTGACACTGGTTGGAA AGTCGGTGAAGGTGCAGGCGCACTGGTGCTAACTGCTG AAAATTCAAATACTAATGCTCTACTTAATAATGCCAATA GCAACAGCTATGGTCACATCAGCGGCCAAGTATTTGGC GCGATTTGTGACATGCAAGGTAACAGCAACACAGCGCGT ATTTGCGATGACTTACTAACCCAAGCCAAGGTGAATAG CAGCCAGATTAGCTTGATTGAAACCAGTATTGCGGTTGA GCAACTTGCCGATTCAGAGCTGGTACTCAATACCCTGC TGCCGAGTGTGAACCAGCGCAGCCAAGCCGCTGATACCC TAGGCCACAATTTGCCGCAGCGGGAATGGCGAGTATTT TGAGCGCCCTGCTTCAGCTTAAAAATCAAGGGCAATTAA AAAACCAAGCACAGCAACAAGCTAATCAAGTGCAGCAC GCGCTCGTTGCCACGTTTAGCCAAGGTAAATGCTCGCAG TTATTGCTCAGTCAAAGTGCGACGCAAGCACACAGTTT GCAGCAAAGGCTTGAACAAGACTTAACGCTTTCTGAGCA AAAACACTTAATTAAACAAGTGACACTTGGTGGCCGCG ATATCTATCAGCATATCCTTGATACGCCGTTGGCGGACA TAGATGCAATAAAGCAAAAAGCCCAAGCCATCACTGCA TTGCCAAAGCGCAGCCAACGCAAACATTTGGCCCAAATA GCGAGCAAAGACACGAGTGGCTTTGCAACAAGCAGCCC AACCACGGCTCTACAAAAAGAGACATTAAGCAGCATGCC AATTAATGCCCTAAGCACGCCCAATGACAACGCAGCTC AAACAGAGCTAAAAGACGCTGCATTTATACGTAATCAGC AACTCGCCCGTGAAGCCCATTTAGCCTTTTTACAAAGC CGTGCGCAGGGCCTGAAACTGGCCGATGCCTTGATGAAA GCCCAGCTTGCCAGCGAGTTAGCCGTCAATGGCCAAGC GACGCCGGTACAACAGCAAGCCACTGTTCAGGCGCCAGT GTATGCATCTGCTCATACACCTGAGCTAGCTCCAGTTG TTAACTCAGAGGCAAACCCAGCTGCGCTTTATCCAAACC ATGCAAAAGTGCCTCTGTACACGCCGCCAACACCGATA AGCAAGCCTTGCATTTGGGATTATGCGGATTTAGTCGAG TACGCCGAAGGCGATATTGCTAACGTCTTTGGCCAAGA TTACGCCATTATCGACAGCTATTCGCGCCGTGTACGCCT GCCAACGACTGACTATTTGCTGGTATCTCGGGTAACGA AATTAAACGCCCAGATGAACCAATATCAGCCTTGCACTA TGACCACAGAATACGACATTCCTGTGGACGCGCCGTAT TTGGTCGATGGCCAAATTCCTTGGGCCGTAGCGGTTGAA TCGGGTCAATGCGACTTGATGCTGATCAGCTACTTAGG TATCGACTTTGAAAACAAGGGCGAGCGCGTTTATCGCCT GCTCGATTGCACCCTGACCTTCCTTGGCGACTTACCGC GCGGTGGCGACACCCTGCGCTACGATATTTCAATCAATC ACTTTGCCCGCAATGGCGATACCTTGTTGTTCTTCTTC TCCTACGAATGTTTCGTGGGCGACAAGCTGATCCTGAAA ATGGATGGCGGCTGTGCCGGCTTCTTCACCGATAAAGA ACTAGCCGACGGCAAAGGCGTTATTCACACCGAAGCCGA AATCAAAGCGCGCAACCTCGCCTTGAACAATCCGAATA AGCCGCGCTTTAATCCGTTACTCAACTGCGCGCAAAACC AATTTGATTACAGCCAAATCCATAAACTGCTCGGCGCC GATATCGGTGGCTGTTTTGGCGGCGCACACGCGGCGCAT CAAGCCCAATATGGTTTGCAGCCCTCTTTATGTTTTGC ATCTGAAAAATTCCTGATGATTGAACAAGTCAGCAATCT TGAGGTGCATGGCGGCGCGTGGGGCTTAGGCTCAGTTC AAGGCCATAAGCAGCTCGAAGCCGATCATTGGTATTTCC CGTGTCATTTCAAGGGCGACCAAGTGATGGCGGGGTCG TTAATGGCCGAAGGCTGTGGTCAATTACTGCAATTCTTT ATGCTACATATTGGTATGCACCTCGGTGTTAAAGATGG TCGTTTCCAACCGCTCGAAAACGCGTCACAAAAAGTGCG TTGTCGCGGTCAAGTGTTGCCGCAATCAGGCCTGCTCA CCTATCGTATGGAAATCACTGAAATCGGTATGAGCCCGC GCCCGTATGCTAAGGCGAATATCGATATTCTGCTCAAT GGTAAAGTGGTTGTGGACTTCCAAAACCTTGGGGTGATG ATCAAAGAAGAAGCCGAATGCACCCGCTACCTTGCGGA TAATGATGCCAGCACAGCTGACAATACGACTAAAAATGC TGCCAAAAATGCTGCTTCGGCTGTGCCGCTAGTTTCGA CAACACCCGCATCGTTCGCCGCGCCGTTGATGGCCCAGC TGCCAGATTTAACTGCGCCAACCAATAAAGGCGTAGTG CCGCTTAAGCATGTGCCTGCGCCGATTGCGCAAACGGAT TCAAAGTACGCCAACCGCGTGCCCGATACCCTGCCGTT CACGCCGTACCACATGTTCGAATTTGCCACGGGCGATAT CGAAAACTGCTTCGGCCCCGATTTCAGCATCTATCGCG GCCTTATTCCACCGCGCACGCCTTGCGGTGATTTACAGC TTACCACCCGCGTCATTGCGATTGACGGCAAACGCGGC GAGCTGAAAAAGCCTTCTTCGTGTATCGCCGAATACGAA GTGCCCGCAAACGCTTGGTATTACGATAAAAACAGTCA TCATGCTGTGATGCCCTATTCAGTGCTAATGGAAATATC ACTGCAGCCAAATGGCTTTATTTCAGGCTATATGGGCA CCACCTTGGGCTTCCCCGGCCAAGAGCTGTTTTTCCGTA ACTTAGACGGCAGCGGTAAGCTGCTGCGCCACGTGGAT TTACGCGGCAAAACCATAGTGAACGACTCACGTTTGTTA TCGACTGTGATTGCCGGCAGCAACATCATCCAGAATTT CAGCTTCGAGTTAAGCTGCGATGGCGAGCCTTTCTACCA AGGTAAAGCGGTATTTGGTTACTTCAAGGGCGATGCGC TGAAAAACCAACTCGGCATAGACAACGGCAAGATCACAC AGCCTTGGCATGTGCAAAATGGCATAGCCGCCGATAGC CAAATCAATCTGTTAGATAAACAGCATCGCAGCTTTAAC GCGCCAGAAGGTCAGCCGCATTACCGTTTAGCGGGCGG TCAGCTTAACTTTATCGACAAGGCCGACATAGTGAAAGC CGGCGGTAAAGCGGGCCTTGGCTATTTATACGCCGAGC GCACCATTGACCCGAGTGATTGGTTCTTCCAATTCCACT TCCATCAAGATCCGGTAATGCCAGGCTCATTAGGGGTT GAAGCGATTATCGAGCTGATGCAAACCTATGCGATTGAC CAAGACCTTGGTGCGGGCTTTAAGAGTCCAAAATTCGG CCAGATATTATCGGATATCAAATGGAAGTATCGCGGCCA AATCAACCCATTAAACAAACAGATGTCGCTGGATGTGC ACATTACCAGCGTGACAGACGACAATGGCAAACGCATCA TTATGGGCGATGCCAACTTGAGTAAAGATGGTCTGCGA ATTTATGAAGTCAAAGATATCGCCATCTGTATTGAAGAA GCTTAATCACCTTGCTCATTTAGAAAGACGCTAATTAC AAATACTGTGTCGCCTTGGCCAGAATGCCTAAGGCGGCA ATAAAAAGAGAATACATATGACAAGCCATACTCTCGAT CAATTTAATAGTAATAACGAAAAACTCAGCCCTTGGCCG TGGCAAGTCAACGATGCCGCGCTGAGCTTTGATATCGA CTCATTAGGCAAAAAACTCAAAGATTTAAGCCAAGCCTG TTACTTAGTGAATCACAGTGAAAAAGGCTTAGGCATAG CGCAAACAGCCGAAGTAACCACAAGCGACAGCCAAGCGC CACTAGGCTCACACCCCGTCAGCGCCTTTGCGCCCGCC CTTGGCACCCAAAGTTTAGGCGACAGTAATTTTCGCCGC GTACACGGGGTTAAATACGCTTACTACGCTGGCGCTAT GGCTAACGGTATTGCCTCAGAAGAACTGGTTATCGCGCT GGGCCAAGCGGGCATTTTGTGTTCGTTTGGCGCGGCGG GGTTAATCCCATCCCGCGTTGAAGCGGCCATTACTCGCA TTCAAGCGGCGCTGCCTAATGGTCCTTACGCCTTTAAT TTAATTCACAGCCCAAGCGAGCCCGCATTAGAGCGCGGC AGTGTTGAGTTGTTCTTAAAACATAAAGTGCGCACGGT CGAAGCCTCGGCATTTTTAGGTTTAACGCCACAAATCGT CTATTACCGCGCAGCAGGTTTGAGCCGCGACGCACATG GCGACATCGTCATTGGCAACAAAGTCATAGCCAAAATCA GTCGCACCGAAGTCGCGACTAAGTTTATGGAGCCGGCG CCTGCCAAAATTCTGCAGCAATTAGTCAGTGAAGGCCTT ATCAGCCAAGATCAAATGGCGATGGCGCAACTTGTACC CATGGCGGACGATATCACGGCCGAAGCCGATTCTGGCGG CCATACCGACAATCGTCCACTGGTCACGCTATTGCCGA CGATTTTGGCGCTCAAAGATGAAATCCAAGCTAAGTATC AATACAAGACGCCCATCCGTGTGGGAGCAGGCGGCGGC GTTGGTACCCCCGACGCAGCATTAGCCACCTTCAACATG GGCGCGGCCTTTATCGTCACAGGTTCAATCAACCAAGC GTGTGTGGAGGCGGGCGCGAGCGAACACACACGTAAGTT ACTCGCCACCACAGAAATGGCCGATGTGACTATGGCAC CCGCCGCCGATATGTTCGAAATGGGCGTGAAATTACAAG TGGTTAAGCGTGGCACTCTGTTCCCGATGCGCGCCAAT AAGCTTTATGAAATCTACACCCGTTACGATTCAATTGAT GCCATCCCCGCGGACGAGCGTAAAAAGCTCGAAGAGCA AGTGTTTCGCGCATCACTCGATGACATTTGGGCGGGCAC TGTCGCCCACTTTAACGAGCGCGATCCTAAGCAAATCG AGCGCGCACTGGATAACCCCAAACGTAAGATGGCGTTGA TTTTCCGCTGGTATTTGGGTCTGTCTAGCCGCTGGTCG AACACAGGTGAAGTCGGCCGCGAAATGGATTACCAAATT TGGGCAGGCCCTGCCCTTGGCGCATTTAATGCGTGGGC AAAAGGCAGTTATTTAGATGACTATAAAGCCCGTAATGC GGTCGATTTAGCCAAACATTTAATGGTGGGCGCGGCCT ATCAATCCCGGATTAACTTGCTGTTATCCCAAGGGGTTA GCATTCCAGTTAGCCTGCAACGCTGGAAACCGCTAAAT CGTTTTTAACCCCTAACGGACTATCGAGACATCAAGTAG GGACTCAACTCCCTACTTGATGCAATCTTCTTCACTAC CTCGTTAAATCGAAGCCGCCGACCTTATTACCAATCTAA TCCCTTGTTTGTGCCAATGCCGCTAAGCACTGTCGCTC CAGTGGCACGCCGTAAACCCATCCTTGGGGGCTCAACGG AAAAGTCCCTTTTTCCGATGG (SEQ ID NO: 1) pfaA gene sequence SEQ ID NO: 2 ATGAGCCATACCCCTTCTGTACCTAATTCTGCGACTGAG TCAAAAAAAGATAAACGACTCAACAAACGTTTGAAAGA TATGCCCGTTGCCATCGTCGGCATGGCCAGTATCTTCGC CAACTCGCGTTATTTAAATAAATTTTGGGATTTGATCA GCGAAAAAATCGATGCCATTACTGACATCCCAGACACCC ATTGGCGCGCCGAAGATTACTACGATGCAGACAAAAGC AAAGCCGATAAAAGCTACTGTAAACGCGGTGGTTTTTTG CCTGAAGTCGACTTCAACCCAATGGAATTTGGTCTGCC GCCCAATATTCTTGAGCTAACAGATACCTCGCAACTGCT GTCGCTGATTGTCGCCAAAGAAGTGCTCGCCGATGCCA ATCTGCCCACCGATTATGATCGCGATCGTATCGGCATCA CGCTAGGCGTGGGCGGTGGTCAAAAAATCAGTCAGAGC TTGAACTCGCGCCTGCAATATCCAGTATTGAAAAAAGTA TTCAAAAGCAGCGGCCTGAGCGATGAAGACAGCGAAAT GCTGATCAAAAAGTTCCAAGACCAATATATCCACTGGGA AGAAAACTCTTTCCCAGGGTCCTTAGGCAACGTGATTG CAGGCCGTATCGCCAACCGTTTTGATTTTGGCGGCATGA ACTGTGTGGTCGATGCTGCCTGCGCGGGCTCACTTGCC GCTATGCGTATGGCGTTGACTGAACTGACCGAAGGTCGC AGCGACATGATGATCACCGGCGGCGTCTGTACCGACAA CTCGCCGTCCATGTATATGAGCTTCTCAAAAACGCCTGC CTTCACTACCAATGAAACCATTCAACCCTTTGATATCG ATTCAAAGGGCATGATGATCGGCGAAGGTATTGGCATGG TAGCACTTAAGCGCCTTGAAGATGCCGAGCGCGATGGC GACCGGATTTATGCCGTCATCAAAGGCGTTGGCGCCTCA TCGGACGGTAAATTTAAGAGTATTTATGCGCCGCGCCC TGAAGGCCAAGCTAAAGCATTGGAGCGCGCCTACGACGA CGCGGGTTTTGCCCCGCACAGCATTGGCTTAGTTGAAG CCCATGGCACGGGCACTGCCGCAGGTGATGTGGCCGAAT TCAATGGCTTAAAATCGGTATTTGCCCAAGGCAACGAC ACCAATCAACATATCGCGTTAGGTTCAGTGAAATCCCAA GTGGGCCACACTAAATCAACCGCAGGTACTGCTGGGGT AATCAAAGCCGCGCTGGCGCTGCACCACAAGGTATTACC TGCGACCATTAACGTCAGCAAGCCTAATCCAAAACTGA ATATCGAAAGCTCACCATTCTATTTAAATACCGAAACGC GCCCTTGGCTGCAACGCACTGACGGTACGCCGCGCCGT GCTGGCATAAGTTCCTTTGGTTTTGGCGGCACTAACTTC CATCTCGTATTAGAAGAATACAAACCCGAGCACAGCCG TGACGAGCAATATCGTCAGCGCAGTGTGCCGCAAACGCT ATTATTTGCCGCCGCCAATAAAGCCGCGCTGCTTAGCG AGTTAAAAGCTGCGCTGAGCCAAAGCTTGAATACGAACG CGAATAAGAGCAGCGCTGCGAGCCTGAACGCTATCGCT CAGCAATATCCGCTACGCGCGCTGGCAGACACAGATGCC CGTTTAGGTTTTGTGGCTAAGGATATCGCTCAGCTGCA AGCTCAGTTGAATCAAGCCATCTCTCATCTAGAAAGCAG TGCGCATTTAGAAAGCGGGCTCCTAGATACCAGTGCCA GCGAGGCGTGGCAGTTACCTTCTGGGATCAACTACCGCG CAAAAGCTTTAGTTGCTAAAGATGAATCAAAGAAAGTG GCCGCTCTGTTTGCCGGTCAAGGTTCACAGTACCTGAAC ATGGGCCGCGAACTCGCCTGCCATTTCCCTGAAATGCG CCAACAAGTGATGGCCAGCGATAAGGTATTTGCTCACCA CGGCCAAACGCCGTTATCCAACATCCTTTATCCTATTC CGGCATTCGATGCCGATGCGGTTAAAGCCCAAGAAGCGG CGCTGACCAATACCCTGTTCGCCCAAAGCGCCATAGGC GCGGTTTCAATGGCGCAATATTCACTATTAACTCAGGCA GGTTTTGCCCCAGATATGGTGGCGGGTCACAGCTTTGG TGAATTGTCAGCCCTTTGCGCGGCTGGCGTGATTTCGAG TGTTGATTACGTCGAACTGGCCTTCGCCCGTGGGCACG CCATGGCGCAAGTGCCAAGTGATACTGACGCTCAAGTCG ATTTAGGCAAGATGTTTGCCATCATCCTCAAACAAAAG AGTGATATCGATGCGCTTAATCGCTGTTTAGCTCAGTTT GATGGCGTTAAAATTGCCAACTACAACGCGCCGACACA ATTAGTCATCGCAGGTGGCACAGAGCAAACTCAGCTGGC TGCCAAAGCTATTAGCGAACAGGGCTTTAAAGCGATTG CCCTGCCCGTCTCTGGCGCGTTCCACACCCCCTTGGTTG GGCACGCACAAAAGCCATTTGCCAAGGCCATTGATAAA GCTAAATTCAGCGCGCCTTCAATCGCACTTTATGCCAAC GGCACAGGTCAATTGCACCCAAGCGATGGCAAAGCGAT TAAAGCAGAATTCAAACAACACATGCTGCAATCGGTTCG TTTTAGCGAACAACTGCAGGCCATGTATGATGCTGGCG CCCGTGTGTTTGTTGAGTTTGGCCCTAAGAACATACTAC AAAAGCTCGTTGAAAATACCTTAAGTGAACACTTAAAT GAGCTTTGCCTTGTCAGCATGAACCCGAACCCTAAGGGC GACAGTGACAGTCAATTACGCTTAGCCGCTGTGCAACT CGCGGTAGCCGGTGTGGCGTTAACTGAGGTTGACCCCTA TCAAGCCGTCACGTCACAGGAGATTGCCGAGCGTGAAG CGCCATCGGCGATGAACATCAAACTCACCGCCACTAACC ACATCAGTGCCGCAACCCAAGCTAAGATGGCGAAATCT CTCGCCACAGGCAGCGTGACTAGCCAAGTGCAATATGTG GATCGCATCGTTGAAACGATTCTCGAAAAAGAAGTCGA AAAAATCGTTCAGAAAGAAGTCATAGTCGAAAAAGTGGT TGAGAAAATAATAGAAGTGGAAGCGAATCAAGTGGCAG CTGTTGAAATGAAACAAAAACTCCTAAGCGTGACGCAAG GCTTAAATCACCAACAAGCGACGGCGCAGATGAGCCCA AGCACAGCAAACGTCAGCGGCGATGCGTTAACGGCATTT TTCAGCGCACAATCTCAAGCGGCGCAGTTACATCAACA GTTTTTAGCCATCCCGCAGCAATATGGCGATACTTTCAC GACACTGATGACAGAACAAGCCAAGATGGCGAGCCTTG GCATTGCAATCCCAGAAAGCCTGCAACGCTCGATGGAAA TGTTCCACCAGCATCAAGCGCAAACTCTGCAAAGCCAC GCCGAATTTATGCAACTGCAAAGCAGTAGCAGCCAAGCG GCCTTGGCCATGTTAAACAATGCGCCGATTAACTTTAC CCCAGCGGTTGCAAGTCAGCCACAAGCAACTGTACCTGT TGCTCCTGCACCTGTTGCTGCATCGACAGTGGCACACA ATGCCGCACCAGTCGCGGCTCAGGCTGTTGCGACAAGAC CAGCGGTTAGCACACCCGTTCCTCCTGTTGTGCAAACC GCTCCCGTTGCTTATGCGCCTGCTGCGACAGTACAAGTT GCGCCAGCAGCTCCTGCCGCACCCGCATTAGTCATGCC TGCTGTGGTGATGCCAGAAGTGACACCTGTGGCACCAAC AATGAACGGTTTAAGTGCTGAAAAAGTACAGCAAACAA TGATGGCTGTGGTGGCTGGCAAGACAGGCTATCCGACTG AAATGCTGGAGCTCAGCATGGATATGGAAGCCGATCTC GGTATCGATTCAATTAAGCGCGTTGAAATCTTAGGCACA GTGCAAGACGAGCTGCCGAGTTTACCTGAACTCAGCCC AGAAGATTTGGCTGAGTGCCGTACCTTGGGTGAAATCGT AAGCTATATGAATAGCAAATTACCTGCAGCGGGAACTA TTGCTTCGACAGCAGCGCCAGTTGCTCAAACCATTGCCG CTCCTGCATCTAACGCTTTAAGCGCTGCATTAGTTCAA CAAACTATGATGGCTGTGGTGGCCGACAAGACAGGCTAC CCCACTGAAATGCTGGAACTTGGCATGGATATGGAAGC CGATTTAGGTATCGATTCTATTAAGCGCGTTGAGATTTT AGGCACAGTGCAAGACGAACTGCCGGGTTTACCCGAAC TTAGCCCAGAAGATTTGGCCGAATGTCGCACCTTAGGTG AAATCGTTACCTATATGAACTCTAAACTGCCAGCAGCG GGTTCAGTTGTAGTTTCTACAACAGCACAAGCCGCGCCT GCCGACAGCGGTTTAAGCGCTGAGTTAGTGCATTCTTC TGAAATCGAGAGCACCATGATGGCTGTGGTGGCCGACAA GACAGGCTATCCGACTGAAATGCTAGAACTGAGCATGG ATATGGAAGCCGATCTCGGTATCGACTCCATCAAGCGCG TTGAAATCCTAGGCACAGTGCAAGACGAACTGCCGGGC TTACCTGAACTTAGCCCAGAAGATTTGGCCGAGTGTCGT ACCTTGGGTGAAATCGTTAGCTATATGAATAGCAAATT ACCAGCAGCAGGAGTTATTGCTTCGACAGCTGCGCCAGT TGCTCAAACCATTGCAGCTCCTGCCGCAATCGGTTTAA GCGCTGCATTAGTGCAACAAACTATGATGGCTGTGGTGG CTGACAAGACAGGTTACCCCACTGAAATGCTGGAACTC AGCATGGATATGGAAGCCGATTTAGGTATCGATTCTATC AAGCGCGTTGAAATTTTAGGCACAGTGCAAGACGAACT TCCTGGCTTACCTGAACTCAATCCTGAAGATCTAGCCGA GTGTCGCACCTTAGGTGAAATCGTTGACTACATGAACA GCAAACTACCTGCGGTTGGCTCGACTTCAACTATAACAG CAGCACAGGTCACTGTTACCGCTACAATAAATAACGGC TTAAGTGCTGAAAAAATCCAACAAACCATGATGTCAGTA GTGGCCGACAAGACGGGCTACCCCACTGAAATGCTGGA GCTCAGCATGGATATGGAAGCCGATTTAGGTATCGATTC TATCAAGCGCGTTGAAATCCTAGGCACAGTACAAGACG AACTGCCGGGTTTACCTGAGCTAAATCCTGAAGATCTGG CCGAGTGTCGCACCTTAGGTGAAATCGTTATGCTCTTT TCGCAAGATTTAGGGCAAGGCAAGCTAGACCAGAATAAT GCGGCGAATGTGGTAAACGTCGCTGTCGCAGTTGAACC GACTATCGATTTGCTACCCCATGATTTACCACAACATGA TTTACCACCACACAGTGAGGTGGTGCTAAAAAAGTTGC CAGCGGCGGCTGAGCTAACGCAACTATCGCCACAGCAAT CGTCAAAGCAATCAGCGCAACAAGCTCAAACACGCGTT TTTGCTAAAGATGCCTGCATTATTATTAGCGATGATGGT CACAATGCTGGCGTACTTGCCGAGAAATTGCATGCTCA AGGTCTAACGGTTGCGGTTGTGCGCTCGCCAGAAAGCCT TGTAGCCAGTGCATCACCGCTCAATAGCCACATTGCCA GCTTCACATTGGCGGCGATTGACGATATCAGCATCAGCG TAGTAATTAATGAGATCAAAACCTTAGGTCAAGTTGCA GGCTTTATTCATCTGCAACCACAACATAAAACCTCAGCC GATGCGAAAGGTTTAGTGTTGTCCAGTGCCGCCAAAGC GTCGGTCGAGCAAGCGTTCTTGTTCGCCAAGCACTTACA GCCGCTTTTAACAACAGCAGCAATCGCCAATACTGGCA GCAGCTTTATCAGCGTCAGTCGTATCGACGGTGGTTTTG GTTATCTTAACCACAGCCAAATTGCCCGCAGCGAGTTA AACCAAGCGGCATTGGCAGGTCTGACTAAAACCTTGAGC CACGAGTGGCCAAGCGTGCATTGCCGCGCCTTAGATAT CGCGCCAGCACTTGATGCCAAGCAACTGGCCAATGCGGT CATAGCTGAGTTATTCGCAACCGATAAGCTTTTAGAAG TCGGAGTGAGTGAAAGTGGTGTGAGTGAAGCTGGCGCAA CTGAAACGCTGGCACGCGTGACCTTAGTTGCAGGCAAG GCAGATACCCGCCACGGCGCAGCTAACTTGACCAGTGCT GATAAAATTCTAGTCACTGGCGGCGCAAAAGGCGTGAC CTTTGAATGTGCCTTGAGTCTGGCAAAACGCAGCAAGGC GCACTTTATCCTTGCCGGCAGAAGCAGCCAACAAACCA TTCCCGCTTGGGCACAAGGTAAAAATAACAGCGAACTTA AAGCCGCAGCTATTGCCCATATTCAAAACTTGGGCGAG AAACCTACACCAAAACAAGTAGATGCCTTAGTATGGCCA GTTCAAAGCAGCCTAGAAATTGCAGCCGCACTAGAAGC CTTTACTGCAGTAGGCGCGAGTGCCGAGTATTTAAGTCT CGATGTAAACAATCCAGACGCGATCGCCAGCACGATAG TGCCGATCACCGAGTTATCACCTATTACCGGCATCATCC ATGGTGCGGGCGTACTCGCCGATAAACACATCCAAGAC AAAACCTTAGATGAGTTAGCACGGGTGTATGGCACTAAG GTCACAGGCATTAGCAATCTGCTTGCCGCACTGGATTT AGATAAAGTAAAACTGATTGCCCTGTTCTCTTCGGCGGC GGGTTTTTACGGTAATACCGGCCAAAGCGATTACGCCA TGTCCAACGACATACTCAACAAAGCCGCACTGCAACTGG CGCAGCAACTGCCAAATGCCAAAGTGATGAGCTTCGAT TGGGGTCCGTGGGACGGCGGCATGGTGAATCCTGCGCTG AAAAAGATGTTTATGGATCGCGGTGTTTATGTAATCCC GCTAAAAGCAGGTGCTGAGTTATTTGCGAGCCAATTACT GAGCAACACAGGCGCACAATTGCTGGTCGGTACTGACA TGCAAGGCAGCGCACCTCATGACGACACGCCTAACGAAG TACAAGAAACTGAAGGTAGTAATCTAAAAAAGCCTGAA GCGGATCTGACCACTGACTCGTCGGATCCGCATGCTTTG CTTAACGCTTTAAATCCAAGCGCAGTAAATATAAGCGC TGTAAAACTGCAGCGCACACTGGACCCTAAGGCAATGAT CTTTATTGAAGATCACTGCATTAACGGTAATCCCGTAT TACCGACTGTGTGCGCGATTCAGTGGATGCGTGAAGCCG CCTTTGATGTGCTCAAGCAACCAGTCAAAGTGCAAAGC TACAAGCTGTTGAAGGGCATTATTTTCGACGCTATGACG TTAGAAAACGGCGCGCCCATCACGCTAGAACTTGAGCT TGCACCGATTGCGTTAACGGATAAAGCCGCGAAAGACAC AGACGAGTACTTGAGTGGACAATTCAGTGCCTTAATCA GCTTTGAAGGTCGTCCGCAATATCAAGCCATCTTAGTGA TTGATGATGCTCCTAGTGATAACTTAGCCACTAATAGC AAAGCAACTGCCTTTGACGCGCATAGCTTGGCAGGATTT TCTGCCATCACAACCGCAAGCAGCCTTTACAGCGACGG CACGCTTTTCCACGGCCCAAGACTGCAAGGTATTGAGTC TGTGGTGAAGTTTGACGATGCGAGCTTAGTCGCCAAGG TGAGTCTTCCCCATGTTGCGTTAGCGGACTGCGGGAGTT TTGTGCCCAATCTGGCCCCTAAAGGCTCACAAGCTTTT GCAGAAGATTTGTTGCTGCAGGCGATGCTGGTATGGGCA AGACTCAAATATGGCGCGGCGAGTTTGCCATCAAGCAT TGGTGAGTTTATCTCCCATGCGCCGTTTGCCTTTGGCGA TACAGGGTATCTAGTGCTCGAGGTGGTAAAACACTCTG GCCGTGCCCTTGAAGCCAATATTGCACTTTATCATCAAG ATGGCCGCTTGAGCTGCGAGATGAACAACGCCAAAGTC ACCATCAGCAAAAACCTTAACGGTGCATTCTTAGCGAAT AAAGTTGCTAATAAAAGCATTGAAAGCGTGGAGGCAAA AGTCGAGT (SEQ ID NO: 2) pfaB gene sequence SEQ ID NO: 3 ATGACCCATAACGGCCAAGCTGAAACGTCGATAGCTGAC AAGGCTATTGCTGACAAAAGCGCCAAGCCACTGCGTAT TGCAGTGTTGCTTGGCGATGCGGTCAACCTTGACTCTCA CTCAGCCCAAGTATTAGGGACGTTTACTGAACGTGAAA GAGTTCAAATTTGCGCTGCCGATGCCAACCAATCGACAG TACATAGGCCGACAGCTCATGAACCCAAAGAGCATGAA CCGTCAGTGTATGAGCAAAGGTCGCTCACAGCCTTGTTA GGCCAAGCAACAACAGCCATTGAGCAAGGCAAGCTCGT TGAACTTAAATTTGAGGATGGTAATCTACCCCAGTATCT GTATTTGCTCGATGGTTTACGCGCCGCCAAACTGCGTC TTCACGCCCACGCGTTTATTGCTGGCTTTGCCGCTGGCA ATGAGGTTACAGATGTTGCAAATGCGGCGACTGTTGCA AATAAGGCGTTAGCGGCAGCAAAGCGCAGCCCAGCTCAA ACCGTTCAACATCAGACTGTCGCCAACACACTCAACGA GGCATTTGTTGCGCTTCGCCAAGGCGTAACCGCCCTCGC CGCGCGAACACAAGCGCCGCTTAAAGGCACTACTGGTA TAAAACAGACAAACGACACCAATCATCAAACTGGCTATT GGTTTAGCGACCAACATCAAGCGCGGGTGTTGTGTCTA AATCTTGTAGCAAAGACGTCACATCAAGCGGATGAGTCT CGAAATCTAAGCCAAAGCCTAGTGCTGACCCAAGGCAC ACAACTCGCCGCGCCCAAAGCCCTTGTCGATGAAAACCG GCTGTTTGTGCCGATAAGTAGTGACAGTATTAATGAGT TAAAAGCAAAGCTGTTTCAGTTGCTTAGTTCACTAGACA TTGGCGCACCAGACACGTCATCTGCATCACATCAACTC ACATTCTGGTTTGAACGCTACGATGCCAACGCACCACTG GCATTAGTCTTGATGGCGGCATCAAGTGATGATCTCAA ACTTGAAGCCAAAGCCATGCTTACGGCGCTTGAAAATGA TGCTATTCGTCATCATGGTCAACACTTTAAGACACCAG CGGGTAGCTGCTTTACCGCTAAGCCGCTAGGGGATGCGG GACTGACTTTTGTGTATCCTGGCGTTGGCACGGTTTAC GCCAATATGTTCAATAACTTGCATGAGTATTTCCCCGCG CTGTATCACCAGTTAGAACGTGAAGGCGATTTAAGCGC CATGCTGCAATCACCGCAGATTTATGCGGCAGATGTTAA AACCGCGGCTGGCATGAACCTAAGTCAGCAAGCGATTA GTGGCGTGGGGGCCAGTTATCTATTCACTAAACTGTTGA CCCAAGTCTTTAATATTAAGCCTAAAATGGCGCTCGGT TACTCAATGGGCGAAGCAGCCATGTGGGCCAGCCTAGAT GTGTGGCAAACACCGCACGCTATGATTAACGCCACTGA AAATAGCGATATTTTCAACCATGCGATTTCTGGTGAGCT AACTGCTGTGCGCCGAGCATGGCAGCTTGCGGATGATG AAGCCATAGTGTGGAACAGCTTTGTGGTGCGCGCTGATA GCCATGAAATCAAGGCATTATTGCCAGAGTTTCCTCGC GCCTACTTAGCCATCACCCAAGGTGATACTTGTGTCATT GCAGGCTGCGAAACAAGCTGTAAAGCCCTGCTTGCCAC GTTAGGCAAACGCGGGATTGCCGCCAATCGCGTCACGGC AATGCATACCGCGCCTGCCCTGTTAGTCCATGGGCAAG TACAAGATTTCTATACTCAAGCGCTAAAACCTGAGGCAC TGGAACCTGATGCGCTGAAAGCGGCAGCGCAAGATTCG TCTGTTCGCTTTATTAGCGCAGCGCAAACTGCGCCAGTA ATCGTGGATAGCCACAGCATTGGCCGCGCGATTGCCGA TACCTTTTGTTCGCCACTCGACTTTAGTGCGCTCATTCA AAATGCCACTGAGCAAGGCGCAAGGCTGTTTGTCGAAG TGGGCGCCGATAGGCAAACCAGTACACTCATAGATAAAA TCAGCCATGCCCACGCAAGCCAAAGCTCTGCGAACGCG GCGACAGCCGCCATTGCCTGCAATGCCAAAGGTGCCGAC GCGATCACCAGCTTACTCAAGTGTTTAGCTCAGCTAAT AAGCCACAGAGTGCCGCTTTCGCTCACGCCGCTTATTCA GCCATTAAGTGCTAACGCAGCCCCTTTATCATCAGCAG TATCACCAAAAGGAGAACCCCAGT (SEQ ID NO: 3) pfaC gene sequence SEQ ID NO: 4 ATGGCGAGTATTTTGAGCGCCCTGCTTCAGCTTAAAAAT CAAGGGCAATTAAAAAACCAAGCACAGCAACAAGCTAA TCAAGTGCAGCACGCGCTCGTTGCCACGTTTAGCCAAGG TAAATGCTCGCAGTTATTGCTCAGTCAAAGTGCGACGC AAGCACACAGTTTGCAGCAAAGGCTTGAACAAGACTTAA CGCTTTCTGAGCAAAAACACTTAATTAAACAAGTGACA CTTGGTGGCCGCGATATCTATCAGCATATCCTTGATACG CCGTTGGCGGACATAGATGCAATAAAGCAAAAAGCCCA AGCCATCACTGCATTGCCAAAGCGCAGCCAACGCAAACA TTTGGCCCAAATAGCGAGCAAAGACACGAGTGGCTTTG CAACAAGCAGCCCAACCACGGCTCTACAAAAAGAGACAT TAAGCAGCATGCCAATTAATGCCCTAAGCACGCCCAAT GACAACGCAGCTCAAACAGAGCTAAAAGACGCTGCATTT ATACGTAATCAGCAACTCGCCCGTGAAGCCCATTTAGC CTTTTTACAAAGCCGTGCGCAGGGCCTGAAACTGGCCGA TGCCTTGATGAAAGCCCAGCTTGCCAGCGAGTTAGCCG TCAATGGCCAAGCGACGCCGGTACAACAGCAAGCCACTG TTCAGGCGCCAGTGTATGCATCTGCTCATACACCTGAG CTAGCTCCAGTTGTTAACTCAGAGGCAAACCCAGCTGCG CTTTATCCAAACCATGCAAAAGTGCCTCTGTACACGCC GCCAACACCGATAAGCAAGCCTTGCATTTGGGATTATGC GGATTTAGTCGAGTACGCCGAAGGCGATATTGCTAACG TCTTTGGCCAAGATTACGCCATTATCGACAGCTATTCGC GCCGTGTACGCCTGCCAACGACTGACTATTTGCTGGTA TCTCGGGTAACGAAATTAAACGCCCAGATGAACCAATAT CAGCCTTGCACTATGACCACAGAATACGACATTCCTGT GGACGCGCCGTATTTGGTCGATGGCCAAATTCCTTGGGC CGTAGCGGTTGAATCGGGTCAATGCGACTTGATGCTGA TCAGCTACTTAGGTATCGACTTTGAAAACAAGGGCGAGC GCGTTTATCGCCTGCTCGATTGCACCCTGACCTTCCTT GGCGACTTACCGCGCGGTGGCGACACCCTGCGCTACGAT ATTTCAATCAATCACTTTGCCCGCAATGGCGATACCTT GTTGTTCTTCTTCTCCTACGAATGTTTCGTGGGCGACAA GCTGATCCTGAAAATGGATGGCGGCTGTGCCGGCTTCT TCACCGATAAAGAACTAGCCGACGGCAAAGGCGTTATTC ACACCGAAGCCGAAATCAAAGCGCGCAACCTCGCCTTG AACAATCCGAATAAGCCGCGCTTTAATCCGTTACTCAAC TGCGCGCAAAACCAATTTGATTACAGCCAAATCCATAA ACTGCTCGGCGCCGATATCGGTGGCTGTTTTGGCGGCGC ACACGCGGCGCATCAAGCCCAATATGGTTTGCAGCCCT CTTTATGTTTTGCATCTGAAAAATTCCTGATGATTGAAC AAGTCAGCAATCTTGAGGTGCATGGCGGCGCGTGGGGC TTAGGCTCAGTTCAAGGCCATAAGCAGCTCGAAGCCGAT CATTGGTATTTCCCGTGTCATTTCAAGGGCGACCAAGT GATGGCGGGGTCGTTAATGGCCGAAGGCTGTGGTCAATT ACTGCAATTCTTTATGCTACATATTGGTATGCACCTCG GTGTTAAAGATGGTCGTTTCCAACCGCTCGAAAACGCGT CACAAAAAGTGCGTTGTCGCGGTCAAGTGTTGCCGCAA TCAGGCCTGCTCACCTATCGTATGGAAATCACTGAAATC GGTATGAGCCCGCGCCCGTATGCTAAGGCGAATATCGA TATTCTGCTCAATGGTAAAGTGGTTGTGGACTTCCAAAA CCTTGGGGTGATGATCAAAGAAGAAGCCGAATGCACCC GCTACCTTGCGGATAATGATGCCAGCACAGCTGACAATA CGACTAAAAATGCTGCCAAAAATGCTGCTTCGGCTGTG CCGCTAGTTTCGACAACACCCGCATCGTTCGCCGCGCCG TTGATGGCCCAGCTGCCAGATTTAACTGCGCCAACCAA TAAAGGCGTAGTGCCGCTTAAGCATGTGCCTGCGCCGAT TGCGCAAACGGATTCAAAGTACGCCAACCGCGTGCCCG ATACCCTGCCGTTCACGCCGTACCACATGTTCGAATTTG CCACGGGCGATATCGAAAACTGCTTCGGCCCCGATTTC AGCATCTATCGCGGCCTTATTCCACCGCGCACGCCTTGC GGTGATTTACAGCTTACCACCCGCGTCATTGCGATTGA CGGCAAACGCGGCGAGCTGAAAAAGCCTTCTTCGTGTAT CGCCGAATACGAAGTGCCCGCAAACGCTTGGTATTACG ATAAAAACAGTCATCATGCTGTGATGCCCTATTCAGTGC TAATGGAAATATCACTGCAGCCAAATGGCTTTATTTCA GGCTATATGGGCACCACCTTGGGCTTCCCCGGCCAAGAG CTGTTTTTCCGTAACTTAGACGGCAGCGGTAAGCTGCT GCGCCACGTGGATTTACGCGGCAAAACCATAGTGAACGA CTCACGTTTGTTATCGACTGTGATTGCCGGCAGCAACA TCATCCAGAATTTCAGCTTCGAGTTAAGCTGCGATGGCG AGCCTTTCTACCAAGGTAAAGCGGTATTTGGTTACTTC AAGGGCGATGCGCTGAAAAACCAACTCGGCATAGACAAC GGCAAGATCACACAGCCTTGGCATGTGCAAAATGGCAT AGCCGCCGATAGCCAAATCAATCTGTTAGATAAACAGCA TCGCAGCTTTAACGCGCCAGAAGGTCAGCCGCATTACC GTTTAGCGGGCGGTCAGCTTAACTTTATCGACAAGGCCG ACATAGTGAAAGCCGGCGGTAAAGCGGGCCTTGGCTAT TTATACGCCGAGCGCACCATTGACCCGAGTGATTGGTTC TTCCAATTCCACTTCCATCAAGATCCGGTAATGCCAGG CTCATTAGGGGTTGAAGCGATTATCGAGCTGATGCAAAC CTATGCGATTGACCAAGACCTTGGTGCGGGCTTTAAGA GTCCAAAATTCGGCCAGATATTATCGGATATCAAATGGA AGTATCGCGGCCAAATCAACCCATTAAACAAACAGATG TCGCTGGATGTGCACATTACCAGCGTGACAGACGACAAT GGCAAACGCATCATTATGGGCGATGCCAACTTGAGTAA AGATGGTCTGCGAATTTATGAAGTCAAAGATATCGCCAT CTGTATTGAAGAAGCTT (SEQ ID NO: 4) pfaD gene sequence SEQ ID NO: 5 ATGACAAGCCATACTCTCGATCAATTTAATAGTAATAAC GAAAAACTCAGCCCTTGGCCGTGGCAAGTCAACGATGC CGCGCTGAGCTTTGATATCGACTCATTAGGCAAAAAACT CAAAGATTTAAGCCAAGCCTGTTACTTAGTGAATCACA GTGAAAAAGGCTTAGGCATAGCGCAAACAGCCGAAGTAA CCACAAGCGACAGCCAAGCGCCACTAGGCTCACACCCC GTCAGCGCCTTTGCGCCCGCCCTTGGCACCCAAAGTTTA GGCGACAGTAATTTTCGCCGCGTACACGGGGTTAAATA CGCTTACTACGCTGGCGCTATGGCTAACGGTATTGCCTC AGAAGAACTGGTTATCGCGCTGGGCCAAGCGGGCATTT TGTGTTCGTTTGGCGCGGCGGGGTTAATCCCATCCCGCG TTGAAGCGGCCATTACTCGCATTCAAGCGGCGCTGCCT AATGGTCCTTACGCCTTTAATTTAATTCACAGCCCAAGC GAGCCCGCATTAGAGCGCGGCAGTGTTGAGTTGTTCTT AAAACATAAAGTGCGCACGGTCGAAGCCTCGGCATTTTT AGGTTTAACGCCACAAATCGTCTATTACCGCGCAGCAG GTTTGAGCCGCGACGCACATGGCGACATCGTCATTGGCA ACAAAGTCATAGCCAAAATCAGTCGCACCGAAGTCGCG ACTAAGTTTATGGAGCCGGCGCCTGCCAAAATTCTGCAG CAATTAGTCAGTGAAGGCCTTATCAGCCAAGATCAAAT GGCGATGGCGCAACTTGTACCCATGGCGGACGATATCAC GGCCGAAGCCGATTCTGGCGGCCATACCGACAATCGTC CACTGGTCACGCTATTGCCGACGATTTTGGCGCTCAAAG ATGAAATCCAAGCTAAGTATCAATACAAGACGCCCATC CGTGTGGGAGCAGGCGGCGGCGTTGGTACCCCCGACGCA GCATTAGCCACCTTCAACATGGGCGCGGCCTTTATCGT CACAGGTTCAATCAACCAAGCGTGTGTGGAGGCGGGCGC GAGCGAACACACACGTAAGTTACTCGCCACCACAGAAA TGGCCGATGTGACTATGGCACCCGCCGCCGATATGTTCG AAATGGGCGTGAAATTACAAGTGGTTAAGCGTGGCACT CTGTTCCCGATGCGCGCCAATAAGCTTTATGAAATCTAC ACCCGTTACGATTCAATTGATGCCATCCCCGCGGACGA GCGTAAAAAGCTCGAAGAGCAAGTGTTTCGCGCATCACT CGATGACATTTGGGCGGGCACTGTCGCCCACTTTAACG AGCGCGATCCTAAGCAAATCGAGCGCGCACTGGATAACC CCAAACGTAAGATGGCGTTGATTTTCCGCTGGTATTTG GGTCTGTCTAGCCGCTGGTCGAACACAGGTGAAGTCGGC CGCGAAATGGATTACCAAATTTGGGCAGGCCCTGCCCT TGGCGCATTTAATGCGTGGGCAAAAGGCAGTTATTTAGA TGACTATAAAGCCCGTAATGCGGTCGATTTAGCCAAAC ATTTAATGGTGGGCGCGGCCTATCAATCCCGGATTAACT TGCTGTTATCCCAAGGGGTTAGCATTCCAGTTAGCCTG CAACGCTGGAAACCGCTAAATCGTTTTT  (SEQ ID NO: 5) pfaE gene sequence SEQ ID NO: 6 AATAATCAGCAAGTAAGCTCTCTATGCTGCTCAACTGCG CGGTTAGCGTTATGGGAAGCGCGGCATTACCGGCATGC GCTAAGCTAATGGCAAAGCGATACTCCTCGGTCAAACGA CCAAAATGCGTTTGCCAATGCAAGGAGTCAGCTAAATC CAGTGTGTGTATTGGGATTTGTAGCGGGACTTGTAATGG AATATGTGGATAAACATTAAGCATTGAAGCTACGCCGC AGGCTTGCCTCAATCCGGCATCTTGGCATGATAACTCAA GCGATTCTGCCGCCAATTGCTCAAACGGCTTAAGCTCA AAGGCAAAGGATTTGAGCGACTGCGCTAAACCTAAACCA GTCGCCTTAATATAGGACTCCTTTAGCGCCCATAAATC GAAAAAGCGCTGTCGCTGACTCGCTTCATCGGCGAGAGC GAGCAAGGCTGCAGTTTCTTGGGGAGAAAAATAGTGAT TTAGGATTGGATAGATATCCGTCTTCGGCCGCGACCTTT CAATATCCACCCCAAATAAACCAGACTCAACGCCATCA AACTGAGCCACACCAATCAATAACCAATCGCCGCTATGG CTTAGGTTAAACTCGAGACCAGTTTGCTGCCCTAATGT CGCCGTTAAACTCGGTTTGCCCTTAGCGCCATATTCAAA ACACCACTCATGTGGCGATAAATCAGCATAGCGAGATA ACACGGTCCGTAGCGCCGCCCTCACCTGTAACCCCTTTA TTTGCACCTTGGGATCGCGATAACGCCTGACCTTTGCG AGTTCATCATCACTTAACCAAGACTCGGCAAGTGATGTT TGCGCCGCTGAAATATCGTTCAGTGGAATAAAAAATAG ATCCACATTCACGCCGTTTTTGCTAGATGTTAGAGAGTC GGTCATTCGAGAGCCTAGCTGTACAAAGCCCTGCGGCT GATGAGTTATCATTGGCTGGCTTTTTGGAGAGCAGTTAG CAACATTCTTATCTGGTTCATTATTGTCTAGGTTCATC ATT (SEQ ID NO: 6) 

1. A recombinant Escherichia coli Nissle 1917 (EcN) cell or a variant thereof, comprising genes encoding for pfaA, pfaB, pfaC, pfaD and pfaE, wherein the cell produces one or more omega 3 fatty acids.
 2. The recombinant cell of claim 1, wherein the cell comprises a pfaA gene with at least 90% sequence identity to SEQ ID NO: 2, a pfaB gene with at least 90% sequence identity to SEQ ID NO: 3, a pfaC gene with at least 90% sequence identity to SEQ ID NO: 4, a pfaD gene with at least 90% sequence identity to SEQ ID NO: 5 and a pfaE gene with at least 90% sequence identity to SEQ ID NO:
 6. 3. The recombinant cell of claim 1, wherein the cell produces eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA).
 4. The recombinant cell of claim 3, wherein the cell produces EPA.
 5. The recombinant cell of claim 1, wherein the cell is transformed with a vector comprising one or more genes selected from pfaA, pfaB, pfaC, pfaD and pfaE.
 6. The recombinant cell of claim 5, wherein the one or more genes are present in the cell in a copy number greater than one.
 7. The recombinant cell of claim 1, wherein the cell comprises a gene cluster with at least 90% sequence identity to SEQ ID NO: 1, wherein the gene cluster comprises pfaA, pfaB, pfaC, pfaD and pfaE.
 8. The recombinant cell of claim 1, wherein the cell comprises a vector comprising a gene cluster comprising the nucleic acid sequence of SEQ ID NO:1.
 9. The recombinant cell of claim 1, wherein the EcN variant comprises a genome with at least 90% sequence identity to the EcN genome deposited under GenBank under accession no. CAPM00000000.
 10. (canceled)
 11. The recombinant cell of claim 1, wherein the cell produces more eicosapentaenoic acid (EPA) relative to wild type S. baltica MAC1 when cultured at 15° C.
 12. (canceled)
 13. The recombinant cell of claim 1, wherein the cell produces at least 10 mg of eicosapentaenoic acid (EPA) per gram of cell dry weight (g⁻¹ of CDW) when cultured at 15° C.
 14. A composition comprising the recombinant cell of claim 1 and a pharmaceutically acceptable carrier or a culture media.
 15. The composition of claim 14, further comprising one or more prebiotics or probiotics. 16.-18. (canceled)
 19. A method for producing eicosapentaenoic acid (EPA) in the gastrointestinal tract of a subject comprising orally administering to the subject the recombinant cell of claim
 1. 20. (canceled)
 21. The method of claim 19, wherein the subject is a human or farm animal.
 22. A method for the production of omega 3 unsaturated fatty acids, comprising culturing one or more recombinant cells of claim 1 under conditions suitable for the production of omega 3 unsaturated fatty acids.
 23. The method of claim 22, wherein the recombinant cell is cultured at a temperature between 5° C. and 30° C.
 24. The method of claim 22, wherein the recombinant cell is cultured at a temperature between 10° C. and 25° C.
 25. (canceled)
 26. (canceled)
 27. The method of claim 22, wherein the one or more omega 3 unsaturated fatty acids comprises eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA).
 28. The method of claim 22, further comprising isolating the one or more omega 3 unsaturated fatty acids from the cell culture. 29.-33. (canceled) 